Il-17 homologous polypeptides and therapeutic uses thereof

ABSTRACT

The present invention is directed to novel polypeptides having sequence identity with IL-17, IL-17 receptors and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention. Further provided herein are methods for treating degenerative cartilaginous disorders and other inflammatory diseases.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/549,497, filed Aug. 28, 2009, which is a continuation of U.S.application Ser. No. 10/617,573, filed Jul. 11, 2003, now issued as U.S.Pat. No. 7,771,719 on Aug. 10, 2010, which is a continuation of U.S.application Ser. No. 10/000,157, filed Oct. 20, 2001, now abandoned;which is a continuation-in-part of U.S. application Ser. No. 09/931,836,filed Aug. 16, 2001, now issued as U.S. Pat. No. 7,435,793 on Oct. 14,2008; which is a continuation-in-part of U.S. application Ser. No.09/929,404, filed Aug. 13, 2001, now abandoned; which is acontinuation-in-part of U.S. application Ser. No. 09/918,585, filed Jul.30, 2001, now abandoned; which is a continuation-in-part of U.S.application Ser. No. 09/908,827, filed Jul. 18, 2001, now abandoned;which is a continuation-in-part of U.S. application Ser. No. 09/747,259,filed Dec. 20, 2000, now issued as U.S. Pat. No. 6,569,645 on May 27,2003; which claims priority from provisional application 60/175,481,filed Jan. 11, 2000; and where U.S. application Ser. No. 09/908,827 is acontinuation-in-part of PCT/US01/21735, filed Jul. 9, 2001; which is acontinuation-in-part of PCT/US01/21066, filed Jun. 29, 2001; which is acontinuation-in-part of PCT/US01/19692, filed Jun. 20, 2001; which is acontinuation-in-part of U.S. application Ser. No. 09/874,503, nowabandoned, filed Jun. 5, 2001; which is a continuation-in-part ofPCT/US01/17800, filed Jun. 1, 2001; which is a continuation-in-part ofboth U.S. application Ser. No. 09/854,280, now issued as U.S. Pat No.7,115,398 on Oct. 3, 2006, and U.S. application Ser. No. 09/854,208, nowissued as U.S. Pat No. 7,217,412 on May 15, 2007, both of which werefiled May 10, 2001; which are both continuations-in-part of U.S.application Ser. No. 09/816,744, filed Mar. 22, 2001, now issued as U.S.Pat. No. 6,579,520 on Jun. 17, 2003; which is a continuation-in-part ofPCT/US01/06520, filed Feb. 28, 2001; which is a continuation-in-part ofboth PCT/US00/34956, and U.S. application Ser. No. 09/747,259, bothfiled Dec. 20, 2000; which are both continuations-in-part ofPCT/US00/30873, filed Nov. 10, 2000; which is a continuation-in-part ofPCT/US00/23328, filed Aug. 24, 2000.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides having sequence similarity to interleukin-17 and tointerleukin-17 receptor protein, designated herein as “PRO”polypeptides.

BACKGROUND OF THE INVENTION

Extracellular proteins play important roles in, among other things, theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, including aspharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents.

Membrane-bound proteins and receptors can play important roles in, amongother things, the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Similarly to secreted proteins, membrane-bound proteins and receptormolecules have various industrial applications, including aspharmaceutical and diagnostic agents. Receptor immunoadhesins, forinstance, can be employed as therapeutic agents to block receptor-ligandinteractions. The membrane-bound proteins can also be employed forscreening of potential peptide or small molecule inhibitors of therelevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native secreted proteins and native receptor or membrane-boundproteins. Many efforts are focused on the screening of mammalianrecombinant DNA libraries to identify the coding sequences for novelsecreted proteins. Examples of screening methods and techniques aredescribed in the literature [see, for example, Klein et al., Proc. Natl.Acad. Sci., 93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].

In this regard, the present invention relates to identifying novelsecreted polypeptides and receptors of the interleukin-17 (IL-17) familywhich have been shown to be related to immune-mediated and inflammatorydisease. Immune related and inflammatory diseases are the manifestationor consequence of fairly complex, often multiple interconnectedbiological pathways which in normal physiology are critical to respondto insult or injury, initiate repair from insult or injury, and mountinnate and acquired defense against foreign organisms. Disease orpathology occurs when these normal physiological pathways causeadditional insult or injury either as directly related to the intensityof the response, as a consequence of abnormal regulation or excessivestimulation, as a reaction to self, or as a combination of these.

Though the genesis of these diseases often involves multi-step pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, etc.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

A central event in both humoral and cell mediated immune responses isthe activation and clonal expansion of helper T cells. Helper T cellactivation is initiated by the interaction of the T cell receptor(TCR)-CD3 complex with an antigen-MHC on the surface of an antigenpresenting cell. This interaction mediates a cascade of biochemicalevents that induce the resting helper T cell to enter a cell cycle (theG0 to G1 transition) and results in the expression of a high affinityreceptor for IL-2 and sometimes IL-4. The activated T cell progressesthrough the cycle proliferating and differentiating into memory cells oreffector cells.

In addition to the signals mediated through the TCR, activation of Tcells involves additional costimulation induced by cytokines released bythe antigen presenting cell or through interactions with membrane boundmolecules on the antigen presenting cell and the T cell. The cytokinesIL-1 and IL-6 have been shown to provide a costimulatory signal. Also,the interaction between the B7 molecule expressed on the surface of anantigen presenting cell and CD28 and CTLA-4 molecules expressed on the Tcell surface effect T cell activation. Activated T cells express anincreased number of cellular adhesion molecules, such as ICAM-1,integrins, VLA-4, LFA-1, CD56, etc.

T-cell proliferation in a mixed lymphocyte culture or mixed lymphocytereaction (MLR) is an established indication of the ability of a compoundto stimulate the immune system. In many immune responses, inflammatorycells infiltrate the site of injury or infection. The migrating cellsmay be neutrophilic, eosinophilic, monocytic or lymphocytic as can bedetermined by histologic examination of the affected tissues. CurrentProtocols in Immunology, ed. John E. Coligan, 1994, John Wiley & Sons,Inc.

Immune related diseases could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

Interleukin-17 (IL-17) has been identified as a cellular ortholog of aprotein encoded by the T lymphotropic Herpes virus Saimiri (HSV) [see,Rouvier et al., J. Immunol., 150(12): 5445-5456 (19993); Yao et al., J.Immunol., 122(12):5483-5486 (1995) and Yao et al., Immunity,3(6):811-821 (1995)]. Subsequent characterization has shown that thisprotein is a potent cytokine that acts to induce proinflammatoryresponses in a wide variety of peripheral tissues. IL-17 is adisulfide-linked homodimeric cytokine of about 32 kDa which issynthesized and secreted only by CD4⁺ activated memory T cells (reviewedin Fossiez et al., Int. Rev. Immunol., 16: 541-551 [1998]).

Despite its restricted tissue distribution, IL-17 exhibits pleitropicbiological activities on various types of cells. IL-17 has been found tostimulate the production of many cytokines. It induces the secretion ofIL-6, IL-8, IL-12, leukemia inhibitory factor (LIF), prostaglandin E2,MCP-1 and G-CSF by adherent cells like fibroblasts, keratinocytes,epithelial and endothelial cells. IL-17 also has the ability to induceICAM-1 surface expression, proliferation of T cells, and growth anddifferentiation of CD34⁺ human progenitors into neutrophils. IL-17 hasalso been implicated in bone metabolism, and has been suggested to playan important role in pathological conditions characterized by thepresence of activated T cells and TNF-α production such as rheumatoidarthritis and loosening of bone implants (Van Bezooijen et al., J. BoneMiner. Res., 14: 1513-1521 [1999]). Activated T cells of synovial tissuederived from rheumatoid arthritis patients were found to secrete higheramounts of IL-17 than those derived from normal individuals orosteoarthritis patients (Chabaud et al., Arthritis Rheum., 42: 963-970[1999]). It was suggested that this proinflammatory cytokine activelycontributes to synovial inflammation in rheumatoid arthritis. Apart fromits proinflammatory role, IL-17 seems to contribute to the pathology ofrheumatoid arthritis by yet another mechanism. For example, IL-17 hasbeen shown to induce the expression of osteoclast differentiation factor(ODF) mRNA in osteoblasts (Kotake et al., J. Clin. Invest., 103:1345-1352 [1999]). ODF stimulates differentiation of progenitor cellsinto osteoclasts, the cells involved in bone resorption. Since the levelof IL-17 is significantly increased in synovial fluid of rheumatoidarthritis patients, it appears that IL-17 induced osteoclast formationplays a crucial role in bone resorption in rheumatoid arthritis. IL-17is also believed to play a key role in certain other autoimmunedisorders such as multiple sclerosis (Matusevicius et al., Mult. Scler.,5: 101-104 [1999]). IL-17 has further been shown, by intracellularsignalling, to stimulate Ca²⁺ influx and a reduction in [cAMP]_(j) inhuman macrophages (Jovanovic et al., J. Immunol., 160:3513 [1998]).Fibroblasts treated with IL-17 induce the activation of NF-κB, [Yao etal., Immunity, 3:811 (1995), Jovanovic et al., supra], while macrophagestreated with it activate NF-κB and mitogen-activated protein kinases(Shalom-Barek et al., J. Biol. Chem., 273:27467 [1998]).

Additionally, IL-17 also shares sequence similarity with mammaliancytokine-like factor 7 that is involved in bone and cartilage growth.Other proteins with which IL-17 polypeptides share sequence similarityare human embryo-derived interleukin-related factor (EDIRF) andinterleukin-20.

Consistent with IL-17's wide-range of effects, the cell surface receptorfor IL-17 has been found to be widely expressed in many tissues and celltypes (Yao et al., Cytokine, 9:794 [1997]). While the amino acidsequence of the human IL-17 receptor (IL-R) (866 amino acids) predicts aprotein with a single transmembrane domain and a long, 525 amino acidintracellular domain, the receptor sequence is unique and is not similarto that of any of the receptors from the cytokine/growth factor receptorfamily. This coupled with the lack of similarity of IL-17 itself toother known proteins indicates that IL-17 and its receptor may be partof a novel family of signalling proteins and receptors. It has beendemonstrated that IL-17 activity is mediated through binding to itsunique cell surface receptor, wherein previous studies have shown thatcontacting T cells with a soluble form of the IL-17 receptor polypeptideinhibited T cell proliferation and IL-2 production induced by PHA,concanavalin A and anti-TCR monoclonal antibody (Yao et al., J.Immunol., 155:5483-5486 [1995]). As such, there is significant interestin identifying and characterizing novel polypeptides having homology tothe known cytokine receptors, specifically IL-17 receptors.

Recently, we have identified two new proteins termed IL-17B and IL-17Cthat are clearly related to IL-17, establishing that there exists afamily of IL-17-like molecules (Li et al., Proc. Natl. Acad. Sci. (USA),97(2):773-778 [2000]). Interestingly, they do not appear to be ligandsfor IL-17 receptor, suggesting that there exists other molecules thatserve as cognate receptors for these factors. Interest in this family ofmolecules has increased as it has become apparent that IL-17 maycontribute to a number of important medical conditions related to immunefunction: including rheumatoid arthritis, immune mediated renaldiseases, hepatobiliary diseases, inflammatory bowel disease, psoriasis,asthma, multiple sclerosis, atherosclerosis, promotion of tumor growth,or degenerative joint disease. Given the potential of IL-17 relatedmolecules to occupy important roles in the control of immune function,there is an interest in the identification of other members of thisfamily and the receptors that direct the actions of these moleculesthrough particular target cell populations. In this respect, the presentinvention describes the cloning and characterization of novel proteins(designated herein as “PRO” polypeptides) that are similar in amino acidsequence to IL-17, and active variants thereof, as well as novelinterleukin-receptor molecules which have been shown to interact withthe novel IL-17 protein ligands.

SUMMARY OF THE INVENTION A. Embodiments

The present invention concerns compositions and methods useful for thediagnosis and treatment of immune related disease in mammals, includinghumans. The present invention is based on the identification of proteins(including agonist and antagonist antibodies) which either stimulate orinhibit the immune response in mammals. Immune related diseases can betreated by suppressing or enhancing the immune response. Molecules thatenhance the immune response stimulate or potentiate the immune responseto an antigen. Molecules which stimulate the immune response can be usedtherapeutically where enhancement of the immune response would bebeneficial. Alternatively, molecules that suppress the immune responseattenuate or reduce the immune response to an antigen (e.g.,neutralizing antibodies) can be used therapeutically where attenuationof the immune response would be beneficial (e.g., inflammation).Accordingly, the PRO polypeptides of the present invention and agonistsand antagonists thereof are also useful to prepare medicines andmedicaments for the treatment of immune-related and inflammatorydiseases. In a specific aspect, such medicines and medicaments comprisea therapeutically effective amount of a PRO polypeptide, agonist orantagonist thereof with a pharmaceutically acceptable carrier.Preferably, the admixture is sterile.

In a further embodiment, the invention concerns a method of identifyingagonists of or antagonists to a PRO polypeptide which comprisescontacting the PRO polypeptide with a candidate molecule and monitoringa biological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native sequence PRO polypeptide. In a specificaspect, the PRO agonist or antagonist is an anti-PRO antibody.

In another embodiment, the invention concerns a composition of mattercomprising a PRO polypeptide or an agonist or antagonist antibody whichbinds the polypeptide in admixture with a carrier or excipient. In oneaspect, the composition comprises a therapeutically effective amount ofthe polypeptide or antibody. In another aspect, when the compositioncomprises an immune stimulating molecule, the composition is useful for:(a) enhancing infiltration of inflammatory cells into a tissue of amammal in need thereof, (b) stimulating or enhancing an immune responsein a mammal in need thereof, (c) increasing the proliferation ofT-lymphocytes in a mammal in need thereof in response to an antigen, (d)stimulating the activity of T-lymphocytes or (e) increasing the vascularpermeability. In a further aspect, when the composition comprises animmune inhibiting molecule, the composition is useful for: (a)decreasing infiltration of inflammatory cells into a tissue of a mammalin need thereof, (b) inhibiting or reducing an immune response in amammal in need thereof, (c) decreasing the activity of T-lymphocytes or(d) decreasing the proliferation of T-lymphocytes in a mammal in needthereof in response to an antigen. In another aspect, the compositioncomprises a further active ingredient, which may, for example, be afurther antibody or a cytotoxic or chemotherapeutic agent. Preferably,the composition is sterile.

In another embodiment, the invention concerns a method of treating animmune related disorder in a mammal in need thereof, comprisingadministering to the mammal a therapeutically effective amount of a PROpolypeptide, an agonist thereof, or an antagonist thereto. In apreferred aspect, the immune related disorder is selected form the groupconsisting of: systemic lupus erythematosis, rheumatoid arthritis,osteoarthritis, juvenile chronic arthritis, spondyloarthropathies,systemic sclerosis, idiopathic inflammatory myopathies, Sjögren'ssyndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia,autoimmune thrombocytopenia, thyroiditis, diabetes mellitus,immune-mediated renal disease, demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious, autoimmune chronic active hepatitis, primary biliarycirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease, gluten-sensitive enteropathy, and Whipple'sdisease, autoimmune or immune-mediated skin diseases including bullousskin diseases, erythema multiforme and contact dermatitis, psoriasis,allergic diseases such as asthma, allergic rhinitis, atopic dermatitis,food hypersensitivity and urticaria, immunologic diseases of the lungsuch as eosinophilic pneumonia, idiopathic pulmonary fibrosis andhypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft -versus-host-disease.

In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody. In one aspect, the presentinvention concerns an isolated antibody which binds a PRO polypeptide.In another aspect, the antibody mimics the activity of a PRO polypeptide(an agonist antibody) or conversely the antibody inhibits or neutralizesthe activity of a PRO polypeptide (an antagonist antibody). In anotheraspect, the antibody is a monoclonal antibody, which preferably hasnonhuman complementarity determining region (CDR) residues and humanframework region (FR) residues. The antibody may be labeled and may beimmobilized on a solid support. In a further aspect, the antibody is anantibody fragment, a monoclonal antibody, a single-chain antibody, or ananti-idiotypic antibody.

In yet another embodiment, the present invention provides a compositioncomprising an anti-PRO antibody in admixture with a pharmaceuticallyacceptable carrier. In one aspect, the composition comprises atherapeutically effective amount of the antibody. Preferably, thecomposition is sterile. The composition may be administered in the formof a liquid pharmaceutical formulation, which may be preserved toachieve extended storage stability. Alternatively, the antibody is amonoclonal antibody, an antibody fragment, a humanized antibody, or asingle-chain antibody.

In a further embodiment, the invention concerns an article ofmanufacture, comprising:

-   (a) a composition of matter comprising a PRO polypeptide or agonist,    antagonist, or an antibody that specifically binds to said    polypeptide thereof;-   (b) a container containing said composition; and-   (c) a label affixed to said container, or a package insert included    in said container referring to the use of said PRO polypeptide or    agonist or antagonist thereof in the treatment of an immune related    disease. The composition may comprise a therapeutically effective    amount of the PRO polypeptide or the agonist or antagonist thereof.

In yet another embodiment, the present invention concerns a method ofdiagnosing an immune related disease in a mammal, comprising detectingthe level of expression of a gene encoding a PRO polypeptide (a) in atest sample of tissue cells obtained from the mammal, and (b) in acontrol sample of known normal tissue cells of the same cell type,wherein a higher or lower expression level in the test sample ascompared to the control sample indicates the presence of immune relateddisease in the mammal from which the test tissue cells were obtained.

In another embodiment, the present invention concerns a method ofdiagnosing an immune disease in a mammal, comprising (a) contacting ananti-PRO antibody with a test sample of tissue cells obtained from themammal, and (b) detecting the formation of a complex between theantibody and a PRO polypeptide, in the test sample; wherein theformation of said complex is indicative of the presence or absence ofsaid disease. The detection may be qualitative or quantitative, and maybe performed in comparison with monitoring the complex formation in acontrol sample of known normal tissue cells of the same cell type. Alarger quantity of complexes formed in the test sample indicates thepresence or absence of an immune disease in the mammal from which thetest tissue cells were obtained. The antibody preferably carries adetectable label. Complex formation can be monitored, for example, bylight microscopy, flow cytometry, fluorimetry, or other techniques knownin the art. The test sample is usually obtained from an individualsuspected of having a deficiency or abnormality of the immune system.

In another embodiment, the invention provides a method for determiningthe presence of a PRO polypeptide in a sample comprising exposing a testsample of cells suspected of containing the PRO polypeptide to ananti-PRO antibody and determining the binding of said antibody to saidcell sample. In a specific aspect, the sample comprises a cell suspectedof containing the PRO polypeptide and the antibody binds to the cell.The antibody is preferably detectably labeled and/or bound to a solidsupport.

In another embodiment, the present invention concerns an immune-relateddisease diagnostic kit, comprising an anti-PRO antibody and a carrier insuitable packaging. The kit preferably contains instructions for usingthe antibody to detect the presence of the PRO polypeptide. Preferablythe carrier is pharmaceutically acceptable.

In another embodiment, the present invention concerns a diagnostic kit,containing an anti-PRO antibody in suitable packaging. The kitpreferably contains instructions for using the antibody to detect thePRO polypeptide.

In another embodiment, the invention provides a method of diagnosing animmune-related disease in a mammal which comprises detecting thepresence or absence or a PRO polypeptide in a test sample of tissuecells obtained from said mammal, wherein the presence or absence of thePRO polypeptide in said test sample is indicative of the presence of animmune-related disease in said mammal.

In another embodiment, the present invention concerns a method foridentifying an agonist of a PRO polypeptide comprising:

-   (a) contacting cells and a test compound to be screened under    conditions suitable for the induction of a cellular response    normally induced by a PRO polypeptide; and (b) determining the    induction of said cellular response to determine if the test    compound is an effective agonist, wherein the induction of said    cellular response is indicative of said test compound being an    effective agonist.

In another embodiment, the invention concerns a method for identifying acompound capable of inhibiting the activity of a PRO polypeptidecomprising contacting a candidate compound with a PRO polypeptide underconditions and for a time sufficient to allow these two components tointeract and determining whether the activity of the PRO polypeptide isinhibited. In a specific aspect, either the candidate compound or thePRO polypeptide is immobilized on a solid support. In another aspect,the non-immobilized component carries a detectable label. In a preferredaspect, this method comprises the steps of:

-   (a) contacting cells and a test compound to be screened in the    presence of a PRO polypeptide under conditions suitable for the    induction of a cellular response normally induced by a PRO    polypeptide; and (b) determining the induction of said cellular    response to determine if the test compound is an effective    antagonist.

In another embodiment, the invention provides a method for identifying acompound that inhibits the expression of a PRO polypeptide in cells thatnormally express the polypeptide, wherein the method comprisescontacting the cells with a test compound and determining whether theexpression of the PRO polypeptide is inhibited. In a preferred aspect,this method comprises the steps of:

-   (a) contacting cells and a test compound to be screened under    conditions suitable for allowing expression of the PRO polypeptide;    and (b) determining the inhibition of expression of said    polypeptide.

In yet another embodiment, the present invention concerns a method fortreating an immune-related disorder in a mammal that suffers therefromcomprising administering to the mammal a nucleic acid molecule thatcodes for either (a) a PRO polypeptide, (b) an agonist of a PROpolypeptide or (c) an antagonist of a PRO polypeptide, wherein saidagonist or antagonist may be an anti-PRO antibody. In a preferredembodiment, the mammal is human. In another preferred embodiment, thenucleic acid is administered via ex vivo gene therapy. In a furtherpreferred embodiment, the nucleic acid is comprised within a vector,more preferably an adenoviral, adeno-associated viral, lentiviral orretroviral vector.

In yet another aspect, the invention provides a recombinant viralparticle comprising a viral vector consisting essentially of a promoter,nucleic acid encoding (a) a PRO polypeptide, (b) an agonist polypeptideof a PRO polypeptide, or (c) an antagonist polypeptide of a PROpolypeptide, and a signal sequence for cellular secretion of thepolypeptide, wherein the viral vector is in association with viralstructural proteins. Preferably, the signal sequence is from a mammal,such as from a native PRO polypeptide.

In a still further embodiment, the invention concerns an ex vivoproducer cell comprising a nucleic acid construct that expressesretroviral structural proteins and also comprises a retroviral vectorconsisting essentially of a promoter, nucleic acid encoding (a) a PROpolypeptide, (b) an agonist polypeptide of a PRO polypeptide or (c) anantagonist polypeptide of a PRO polypeptide, and a signal sequence forcellular secretion of the polypeptide, wherein said producer cellpackages the retroviral vector in association with the structuralproteins to produce recombinant retroviral particles.

In a still further embodiment, the invention provides a method forenhancing the infiltration of inflammatory cells from the vasculatureinto a tissue of a mammal comprising administering to said mammal (a) aPRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) anantagonist of a PRO polypeptide, wherein the infiltration ofinflammatory cells from the vasculature in the mammal is enhanced.

In a still further embodiment, the invention provides a method fordecreasing the infiltration of inflammatory cells from the vasculatureinto a tissue of a mammal comprising administering to said mammal (a) aPRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) anantagonist of a PRO polypeptide, wherein the infiltration ofinflammatory cells from the vasculature in the mammal is decreased.

In a still further embodiment, the invention provides a method ofincreasing the activity of T-lymphocytes in a mammal comprisingadministering to said mammal (a) a PRO polypeptide, (b) an agonist of aPRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein theactivity of T-lymphocytes in the mammal is increased.

In a still further embodiment, the invention provides a method ofdecreasing the activity of

T-lymphocytes in a mammal comprising administering to said mammal (a) aPRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) anantagonist of a PRO polypeptide, wherein the activity of T-lymphocytesin the mammal is decreased.

In a still further embodiment, the invention provides a method ofincreasing the proliferation of T-lymphocytes in a mammal comprisingadministering to said mammal (a) a PRO polypeptide, (b) an agonist of aPRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein theproliferation of T-lymphocytes in the mammal is increased.

In a still further embodiment, the invention provides a method ofdecreasing the proliferation of T-lymphocytes in a mammal comprisingadministering to said mammal (a) a PRO polypeptide, (b) an agonist of aPRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein theproliferation of T-lymphocytes in the mammal is decreased.

In a still further embodiment, the invention provides a method ofstimulating the proliferation of T-cells comprising contacting saidT-cells with a PRO1031 or PRO10272 polypeptide or agonist thereof,wherein said T-cell proliferation is stimulated.

In a still further embodiment, the invention provides a method ofdecreasing the proliferation of T-lymphocytes comprising contacting saidT-lymphocytes with an antagonist of a PRO1031 or PRO10272 polypeptide,wherein the proliferation of T-lymphocytes is decreased.

In a still further embodiment, the invention provides a method ofenhancing the infiltration of inflammatory cells into a tissue of amammal comprising administering an effective amount of a PRO1031polypeptide or agonist thereof, wherein said infiltration is enhanced.

In a still further embodiment, the invention provides a method ofdecreasing the infiltration of inflammatory cells into a tissue of amammal comprising administering an effective amount of an antagonist ofa PRO1031 polypeptide, wherein said infiltration is decreased.

In yet another embodiment, the invention provides a method forinhibiting angiogenesis induced by a PRO1031 polypeptide or an agonistthereof in a mammal comprising administering a therapeutically effectiveamount of an anti-PRO1031 antibody to the mammal. Preferably, the mammalis a human, and more preferably the mammal has a tumor or a retinaldisorder.

In yet another embodiment, the invention provides a method forstimulating angiogenesis induced by a PRO1031 polypeptide in a mammalcomprising administering a therapeutically effective amount of a PRO1031polypeptide or agonist thereof to the mammal. Preferably, the mammal isa human, and more preferably angiogenesis would promote tissueregeneration or wound healing.

In another embodiment, the invention provides a method for inhibitingangiogenesis in a mammal comprising administering a therapeuticallyeffective amount of an antagonist of a PRO1031 polypeptide to themammal, wherein said angiogenesis is inhibited.

In still a further embodiment, the invention concerns the use of aPRO1031, PRO1122, PRO10272, or PRO20110 polypeptide, or an agonist orantagonist thereof as hereinbefore described, or an anti-PRO1031,anti-PRO1122, anti-PRO10272, or anti-PRO20110 antibody, for thepreparation of a medicament useful in the treatment of a condition whichis responsive to the PRO1031, PRO1122, PRO10272, or PRO20110 polypeptideor an agonist or antagonist thereof (e.g., anti-PRO1031, anti-PRO1122,anti-PRO10272, or anti-PRO20110). In a particular aspect, the inventionconcerns the use of a PRO1031, PRO1122, PRO10272, or PRO20110polypeptide, or an agonist or antagonist thereof in a method fortreating a degenerative cartilaginous disorder.

In still a further embodiment, the invention relates to a method oftreating a degenerative cartilaginous disorder in a mammal comprisingadministering a therapeutically effective amount of a PRO1031, PRO1122,PRO10272, or PRO20110 polypeptide, agonist, or antagonist thereof, tosaid mammal suffering from said disorder.

In still a further embodiment, the invention relates to a kit comprisinga composition comprising a PRO1031, PRO1122, PRO10272, or PRO20110polypeptide, or an agonist or antagonist thereof, in admixture with apharmaceutically acceptable carrier; a container containing saidcomposition; and a label affixed to said container, referring to the useof said composition, in the treatment of a degenerative cartilaginousdisorder.

In a further embodiment, the invention relates to a method of detectinga polypeptide designated as A, B, or C in a sample suspected ofcontaining an A, B, or C polypeptide, said method comprising contactingsaid sample with a polypeptide designated herein as D, E, or F anddetermining the formation of a A/D, B/D, C/E or C/F polypeptideconjugate in said sample, wherein the formation of said conjugate isindicative of the presence of an A, B, or C polypeptide in said sampleand wherein A is a PRO1031 polypeptide (herein also designated IL-17B),B is a PRO10272 polypeptide (herein also designated IL-17E), C is aPRO20110 polypeptide (herein also designated IL-17F), D is a PRO5801polypeptide (herein also designated IL-17RH1), E is a PRO1 polypeptide(herein known as IL-17R), and F is a PRO20040 polypeptide (herein alsodesignated IL-17RH2). In one aspect of this embodiment, said samplecomprises cells suspected of expressing said A, B, or C polypeptide.

In another aspect of this embodiment said D, E, or F polypeptide islabeled with a detectable label and said D, E, or F polypeptide isattached to a solid support.

In yet another embodiment, the invention relates to a method ofdetecting a polypeptide designated as D, E, or F in a sample suspectedof containing an D, E, or F polypeptide, said method comprisingcontacting said sample with a polypeptide designated herein as A, B, orC and determining the formation of a A/D, B/D,

C/E, or C/F polypeptide conjugate in said sample, wherein the formationof said conjugate is indicative of the presence of an A, B, or Cpolypeptide in said sample and wherein A is a PRO1031 polypeptide(herein also designated IL-17B), B is a PRO10272 polypeptide (hereinalso designated IL-17E), C is a PRO20110 polypeptide (herein alsodesignated IL-17F), D is a PRO5801 polypeptide (herein also designatedIL-17RH1), E is a PRO1 polypeptide (herein known as IL-17R), and F is aPRO20040 polypeptide (herein also designated IL-17RH2). In one aspect ofthis embodiment, said sample comprises cells suspected of expressingsaid D, E, or F polypeptide. In another aspect of this embodiment, saidA, B, or C polypeptide is labeled with a detectable label and said A, B,or C polypeptide is attached to a solid support.

In still a further embodiment, the invention relates to a method oflinking a bioactive molecule to a cell expressing a polypeptidedesignated as A, B, or C, said method comprising contacting said cellwith a polypeptide designated as D, E, or F that is bound to saidbioactive molecule and allowing said A, B, or C and said D, E, or Fpolypeptides to bind to one another, thereby linking said bioactivemolecules to said cell, wherein A is a PRO1031 polypeptide (herein alsodesignated IL-17B), B is a PRO10272 polypeptide (herein also designatedIL-17E), C is a PRO20110 polypeptide (herein also designated IL-17F), Dis a PRO5801 polypeptide (herein also designated IL-17RH1), E is a PRO1polypeptide (herein known as IL-17R), and F is a PRO20040 polypeptide(herein also designated IL-17RH2). In one aspect of this embodiment,said bioactive molecule is a toxin, a radiolabel or an antibody. Inanother aspect of this embodiment, said bioactive molecule causes thedeath of said cell.

In a further embodiment, the invention relates to a method of linking abioactive molecule to a cell expressing a polypeptide designated as D,E, or F, said method comprising contacting said cell with a polypeptidedesignated as A, B, or C that is bound to said bioactive molecule andallowing said A, B, or C and said D, E, or F polypeptides to bind to oneanother, thereby linking said bioactive molecules to said cell, whereinA is a PRO1031 polypeptide (herein also designated IL-17B), B is aPRO10272 polypeptide (herein also designated IL-17E), C is a PRO20110polypeptide (herein also designated IL-17F), D is a PRO5801 polypeptide(herein also designated IL-17RH1), E is a PRO1 polypeptide (herein knownas IL-17R), and F is a PRO20040 polypeptide (herein also designatedIL-17RH2). In one aspect of this embodiment, said bioactive molecule isa toxin, a radiolabel or an antibody. In another aspect of thisembodiment, said bioactive molecule causes the death of said cell.

In still another embodiment, the invention relates to a method ofmodulating at least one biological activity of a cell expressing apolypeptide designated as A, B, or C, said method comprising contactingsaid cell with a polypeptide designated as D, E, or F or an anti-A,anti-B, or anti-C polypeptide antibody, whereby said D, E, or Fpolypeptide or anti-A, anti-B, or anti-C polypeptide antibody binds tosaid A, B, or C polypeptide, thereby modulating at least one biologicalactivity of said cell, wherein A is a PRO1031 polypeptide (herein alsodesignated IL-17B), B is a PRO10272 polypeptide (herein also designatedIL-17E), C is a PRO20110 polypeptide (herein also designated IL-17F), Dis a PRO5801 polypeptide (herein also designated IL-17RH1), E is a PRO1polypeptide (herein known as IL-17R), and F is a PRO20040 polypeptide(herein also designated IL-17RH2). In one aspect of this embodiment,said cell is killed.

In yet a further embodiment, the invention relates to a method ofmodulating at least one biological activity of a cell expressing apolypeptide designated as D, E, or F, said method comprising contactingsaid cell with a polypeptide designated as A, B, or C or an anti-D,anti-E, or anti-F polypeptide antibody, whereby said A, B, or Cpolypeptide or anti-D, anti-E, or anti-F polypeptide antibody binds tosaid D, E, or F polypeptide, thereby modulating at least one biologicalactivity of said cell, wherein A is a PRO1031 polypeptide (herein alsodesignated IL-17B), B is a PRO10272 polypeptide (herein also designatedIL-17E), C is a PRO20110 polypeptide (herein also designated IL-17F), Dis a PRO5801 polypeptide (herein also designated IL-17RH1), E is a PRO1polypeptide (herein known as IL-17R), and F is a PRO20040 polypeptide(herein also designated IL-17RH2). In one aspect of this embodiment,said cell is killed.

B. Additional Embodiments

In other embodiments of the present invention, the invention provides anisolated nucleic acid molecule comprising a nucleotide sequence thatencodes a PRO polypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80% nucleic acid sequence identity,alternatively at least about 81% nucleic acid sequence identity,alternatively at least about 82% nucleic acid sequence identity,alternatively at least about 83% nucleic acid sequence identity,alternatively at least about 84% nucleic acid sequence identity,alternatively at least about 85% nucleic acid sequence identity,alternatively at least about 86% nucleic acid sequence identity,alternatively at least about 87% nucleic acid sequence identity,alternatively at least about 88% nucleic acid sequence identity,alternatively at least about 89% nucleic acid sequence identity,alternatively at least about 90% nucleic acid sequence identity,alternatively at least about 91% nucleic acid sequence identity,alternatively at least about 92% nucleic acid sequence identity,alternatively at least about 93% nucleic acid sequence identity,alternatively at least about 94% nucleic acid sequence identity,alternatively at least about 95% nucleic acid sequence identity,alternatively at least about 96% nucleic acid sequence identity,alternatively at least about 97% nucleic acid sequence identity,alternatively at least about 98% nucleic acid sequence identity andalternatively at least about 99% nucleic acid sequence identity to (a) aDNA molecule encoding a PRO polypeptide having a full-length amino acidsequence as disclosed herein, an amino acid sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a transmembraneprotein, with or without the signal peptide, as disclosed herein or anyother specifically defined fragment of the full-length amino acidsequence as disclosed herein, or (b) the complement of the DNA moleculeof (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity to (a) a DNA molecule comprising the coding sequence of afull-length PRO polypeptide cDNA as disclosed herein, the codingsequence of a PRO polypeptide lacking the signal peptide as disclosedherein, the coding sequence of an extracellular domain of atransmembrane PRO polypeptide, with or without the signal peptide, asdisclosed herein or the coding sequence of any other specificallydefined fragment of the full-length amino acid sequence as disclosedherein, or (b) the complement of the DNA molecule of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%nucleic acid sequence identity, alternatively at least about 81% nucleicacid sequence identity, alternatively at least about 82% nucleic acidsequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity to (a) a DNA molecule that encodes the same maturepolypeptide encoded by any of the human protein cDNAs deposited with theATCC as disclosed herein, or (b) the complement of the DNA molecule of(a).

Another aspect of the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence encoding a PROpolypeptide which is either transmembrane domain-deleted ortransmembrane domain-inactivated, or is complementary to such encodingnucleotide sequence, wherein the transmembrane domain(s) of suchpolypeptide are disclosed herein. Therefore, soluble extracellulardomains of the herein described PRO polypeptides are contemplated.

Another embodiment is directed to fragments of a PRO polypeptide codingsequence, or the complement thereof, that may find use as, for example,hybridization probes, for encoding fragments of a PRO polypeptide thatmay optionally encode a polypeptide comprising a binding site for ananti-PRO antibody or as antisense oligonucleotide probes. Such nucleicacid fragments are usually at least about 20 nucleotides in length,alternatively at least about 30 nucleotides in length, alternatively atleast about 40 nucleotides in length, alternatively at least about 50nucleotides in length, alternatively at least about 60 nucleotides inlength, alternatively at least about 70 nucleotides in length,alternatively at least about 80 nucleotides in length, alternatively atleast about 90 nucleotides in length, alternatively at least about 100nucleotides in length, alternatively at least about 110 nucleotides inlength, alternatively at least about 120 nucleotides in length,alternatively at least about 130 nucleotides in length, alternatively atleast about 140 nucleotides in length, alternatively at least about 150nucleotides in length, alternatively at least about 160 nucleotides inlength, alternatively at least about 170 nucleotides in length,alternatively at least about 180 nucleotides in length, alternatively atleast about 190 nucleotides in length, alternatively at least about 200nucleotides in length, alternatively at least about 250 nucleotides inlength, alternatively at least about 300 nucleotides in length,alternatively at least about 350 nucleotides in length, alternatively atleast about 400 nucleotides in length, alternatively at least about 450nucleotides in length, alternatively at least about 500 nucleotides inlength, alternatively at least about 600 nucleotides in length,alternatively at least about 700 nucleotides in length, alternatively atleast about 800 nucleotides in length, alternatively at least about 900nucleotides in length and alternatively at least about 1000 nucleotidesin length, wherein in this context the term “about” means the referencednucleotide sequence length plus or minus 10% of that referenced length.It is noted that novel fragments of a PRO polypeptide-encodingnucleotide sequence may be determined in a routine manner by aligningthe PRO polypeptide-encoding nucleotide sequence with other knownnucleotide sequences using any of a number of well known sequencealignment programs and determining which PRO polypeptide-encodingnucleotide sequence fragment(s) are novel. All of such PROpolypeptide-encoding nucleotide sequences are contemplated herein. Alsocontemplated are the PRO polypeptide fragments encoded by thesenucleotide molecule fragments, preferably those PRO polypeptidefragments that comprise a binding site for an anti-PRO antibody.

In another embodiment, the invention provides an isolated PROpolypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

In a certain aspect, the invention concerns an isolated PRO polypeptide,comprising an amino acid sequence having at least about 80% amino acidsequence identity, alternatively at least about 81% amino acid sequenceidentity, alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to a PROpolypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence having at least about 80% amino acidsequence identity, alternatively at least about 81% amino acid sequenceidentity, alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to anamino acid sequence encoded by any of the human protein cDNAs depositedwith the ATCC as disclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence scoring at least about 80% positives,alternatively at least about 81% positives, alternatively at least about82% positives, alternatively at least about 83% positives, alternativelyat least about 84% positives, alternatively at least about 85%positives, alternatively at least about 86% positives, alternatively atleast about 87% positives, alternatively at least about 88% positives,alternatively at least about 89% positives, alternatively at least about90% positives, alternatively at least about 91% positives, alternativelyat least about 92% positives, alternatively at least about 93%positives, alternatively at least about 94% positives, alternatively atleast about 95% positives, alternatively at least about 96% positives,alternatively at least about 97% positives, alternatively at least about98% positives and alternatively at least about 99% positives whencompared with the amino acid sequence of a PRO polypeptide having afull-length amino acid sequence as disclosed herein, an amino acidsequence lacking the signal peptide as disclosed herein, anextracellular domain of a transmembrane protein, with or without thesignal peptide, as disclosed herein or any other specifically definedfragment of the full-length amino acid sequence as disclosed herein.

In a specific aspect, the invention provides an isolated PRO polypeptidewithout the N-terminal signal sequence and/or the initiating methionineand is encoded by a nucleotide sequence that encodes such an amino acidsequence as hereinbefore described. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thePRO polypeptide and recovering the PRO polypeptide from the cellculture.

Another aspect of the invention provides an isolated PRO polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PROpolypeptide and recovering the PRO polypeptide from the cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists to a PRO polypeptide which comprise contactingthe PRO polypeptide with a candidate molecule and monitoring abiological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

In a still further embodiment, the invention concerns a composition ofmatter comprising a PRO polypeptide, or an agonist or antagonist of aPRO polypeptide as herein described, or an anti-PRO antibody, incombination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

Another embodiment of the present invention is directed to the use of aPRO polypeptide, or an agonist or antagonist thereof as hereinbeforedescribed, or an anti-PRO antibody, for the preparation of a medicamentuseful in the treatment of a condition which is responsive to the PROpolypeptide, an agonist or antagonist thereof or an anti-PRO antibody.

In additional embodiments of the present invention, the inventionprovides vectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, yeast, orBaculovirus-infected insect cells. A process for producing any of theherein described polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression of thedesired polypeptide and recovering the desired polypeptide from the cellculture.

In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

In yet another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody.

In yet other embodiments, the invention provides oligonucleotide probesuseful for isolating genomic and cDNA nucleotide sequences or asantisense probes, wherein those probes may be derived from any of theabove or below described nucleotide sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequencePRO1031 cDNA, wherein SEQ ID NO:1 is a clone designated herein as“DNA59294-1381-1”.

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO:1 shown in FIG. 1.

FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a native sequencePRO1122 cDNA, wherein SEQ ID NO:3 is a clone designated herein as“DNA62377-1381-1”.

FIG. 4 shows the amino acid sequence (SEQ ID NO:4) derived from thecoding sequence of SEQ ID NO:3 shown in FIG. 3.

FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a native sequencePRO10272 cDNA, wherein SEQ ID NO:5 is a clone designated herein as“DNA147531-2821”.

FIG. 6 shows the amino acid sequence (SEQ ID NO:6) derived from thecoding sequence of SEQ ID NO:5 shown in FIG. 5.

FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a native sequencePRO21175 cDNA, wherein SEQ ID NO:7 is a clone designated herein as“DNA173894-2947”.

FIG. 8 shows the amino acid sequence (SEQ ID NO:8) derived from thecoding sequence of SEQ ID NO:7 shown in FIG. 7.

FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a native sequencePRO20110 cDNA, wherein SEQ ID NO:9 is a clone designated herein as“DNA166819”.

FIG. 10 shows the amino acid sequence (SEQ ID NO:10) derived from thecoding sequence of SEQ ID NO:9 shown in FIG. 9.

FIG. 11 shows a nucleotide sequence (SEQ ID NO:11) of a native sequencePRO5801 cDNA, wherein SEQ ID NO:11 is a clone designated herein as“DNA115291-2681”.

FIG. 12 shows the amino acid sequence (SEQ ID NO:12) derived from thecoding sequence of SEQ ID NO:11 shown in FIG. 11.

FIG. 13 shows a nucleotide sequence (SEQ ID NO:13) of a native sequencePRO20040 cDNA, wherein SEQ ID NO:13 is a clone designated herein as“DNA164625-2890”.

FIG. 14 shows the amino acid sequence (SEQ ID NO:14) derived from thecoding sequence of SEQ ID NO:13 shown in FIG. 13.

FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a native sequencePRO9877 cDNA, wherein SEQ ID NO:15 is a clone designated herein as“DNA119502-2789”.

FIG. 16 shows the amino acid sequence (SEQ ID NO:16) derived from thecoding sequence of SEQ ID NO:15 shown in FIG. 15.

FIG. 17 shows a nucleotide sequence (SEQ ID NO:17) of a native sequencePRO20026 cDNA, wherein SEQ ID NO:17 is a clone designated herein as“DNA154095-2998”.

FIG. 18 shows the amino acid sequence (SEQ ID NO:18) derived from thecoding sequence of SEQ ID NO:17 shown in FIG. 17.

FIG. 19 shows the alignment of the human IL-17 family members: h-IL17[SEQ ID NO:40]; h-1L17B [PRO1031; SEQ ID NO:2]; h-IL17C [PRO1122; SEQ IDNO:4]; h-IL17D [PRO21175; SEQ ID NO:8]; h-ILE [PRO10272; SEQ ID NO:6];and h-IL17F [PRO20110; SEQ ID NO:10].

FIG. 20 shows the relative tissue expression distribution of the IL-17Bligand (PRO1031).

FIG. 21 shows the relative tissue expression distribution of the IL-17Cligand (PRO1122).

FIG. 22 shows the relative tissue expression distribution of the IL-17Dligand (PRO21175).

FIG. 23 shows mRNA expression of IL-17E (PRO10272) by RT-PCR analysis.RNA from the indicated tissues was subjected to RT-PCR with primers thatwere designed to amplify the entire coding sequence of IL-17E. The PCRproduct was resolved by agarose gel electrophoresis, transferred tonylon membrane and probed with a ³²P-labeled IL-17E cDNA probe.

FIG. 24 shows the relative tissue expression distribution of the IL-17Fligand (PRO20110).

FIG. 25 shows the relative tissue expression distribution of theIL-17RH1 receptor (PRO5801).

FIG. 26 shows the relative tissue expression distribution of theIL-17RH2 receptor (PRO20040).

FIG. 27 shows the relative tissue expression distribution of theIL-17RH3 receptor (PRO9877).

FIG. 28 shows the relative tissue expression distribution of theIL-17RH4 receptor (PRO20026).

FIG. 29 shows immunoprecipitation of IL-17R extracellular domain (ECD)with IL-17, IL-17B (PRO1031) and IL-17C (PRO1122). His-tagged IL-17R ECDwas expressed in 293 cells and metabolically labeled with ³⁵S asdescribed in EXAMPLE 21. The supernatant was recovered and Ni-NTA beadswere used to affinity precipitate the His-tagged IL-17R ECD in thesupernatant (lane 1). In part A., IL-17, IL-17B.Fc and IL-17C.Fc, orcontrol Fc fusion proteins were incubated with the supernatant andprotein-A-agarose beads were added to precipitate the Fc fusionproteins. For the IL-17 immunoprecipitation reaction, anti-IL17antibodies were included. Part B. shows the results of a competitivebinding experiment, wherein immunoprecipitation of IL-17R ECD by IL-17was performed in the presence of a five-fold excess of IL-17B.His andcontrol His-tagged proteins. Precipitates in both part A. and B. wereanalyzed by electrophoresis on NuPAGE (4-12% Bis-Tris) gels. Molecularweight markers are indicated on the left of each panel.

FIG. 30 shows the alignment of the human IL-17 family members (h-IL17(SEQ ID NO:40); H-IL17B [PRO1031; SEQ ID NO:2]; H-IL17C [PRO1122; SEQ IDNO:4]; and H-ILE [PRO10272; SEQ ID NO:6]). The predicted signalsequences are underlined. Conserved cysteines are indicated by bullet,and potential N-linked glycosylation sites are boxed.

FIG. 31 shows mRNA expression of IL-17RH1 receptor (PRO5801). FIG. 31Ashows Northern blot analysis of IL-17RH1 receptor in selected tissues.FIG. 31B shows the quantitative PCR analysis of IL-17RH1 mRNA expressionin selected tissues.

FIG. 32 shows IL-17E (PRO10272) ligand binding to IL-17RH1 receptor(PRO5801). FIG. 32A shows a comparison of IL-17 and IL-17E (PRO10272)ligand binding to IL-17R receptor (herein designated PRO1) and IL-17RH1receptor (PRO5801). 293 cells were transiently co-transfected withexpression vectors for green fluorescent protein (GCP) and IL-17R orIL-17RH1 receptors as indicated. Cells were incubated with IL-17-Fc orIL-17E-Fc protein as indicated and binding was revealed with PEconjugated anti-human Fc antibody. FACS curves show PE staining withinthe co-transfected GFP positive cell population. In FIG. 32B,His-epitope tagged IL-17RH1 receptor extracellular domain was incubatedwith ligand-Fc fusion protein for members of the human IL-17 familydepicted as follows: lane 1, IL-17RH1-His direct load; lane 2, IL-17;lane 3, IL-17B (PRO1031); lane 4, IL-17C (PRO1122); and lane 5, IL-17E(PRO10272). Ligand immunoadhesins were immunoprecipitated with Protein Abeads and bound IL-17RH1 receptor was analyzed by Western Blot analysiswith antibody to the His-epitope tag. The positions of molecular weightmarkers (kDa) are indicated on the left.

FIG. 33 shows the induction of NF-κB by IL-17E (PRO10272). FIG. 33 (partA) shows the results of transiently transfecting human 293 and TK-10cells with the NF-κB responsive luciferase reporter pGL3.ELAM.tk andexpression vector for IL-17E as indicated. Luciferase activity wasdetermined as indicated in EXAMPLE 22. FIG. 33 (part B) depictstitration of NF-κB induction by IL-17E. Human 293 cells were transfectedwith the NF-κB responsive luciferase reporter pGL3.ELAM.tk and theindicated expression vector for IL-17E as indicated.

FIG. 34 shows the effect of IL-17E (PRO10272) on IL-8 production. HumanTK-10 kidney derived cells lines were incubated by Elisa. Shown is thelevel of IL-8 measured minus the level of IL-8 production observed inthe absence of cytokine addition. The experiments were repeated severaltimes with similar results.

FIG. 35 depicts the IL-17 family of cytokines and the complex pattern ofoverlapping receptor-ligand specificities. From left to right, FIG. 35demonstrates that IL-17 ligand binds to the IL-17 receptor (IL-17R;herein designated PRO1); IL-17B ligand (PRO1031) binds to the IL-17RH1receptor (PRO5801); IL-17E ligand (PRO10272) binds to the IL-17RH1receptor (PRO5801); IL-17F ligand (PRO20110) binds to both the IL-17receptor (IL-17R, herein designated PROD as well as to the IL-17RH2receptor (PRO20040); IL-17C ligand (PRO1122) and IL-17D ligand(PRO21175) do not interact with IL-17R, IL-17RH1 or IL-17RH2 receptors.

FIG. 36 depicts bar graphs representing the biological activities ofIL-17, IL-17B (PRO1031), and IL-17C (PRO1122). FIG. 36 (part A.) showshuman foreskin fibroblast (HFF) cells cultured with control Fc fusionprotein, IL-17, IL-17B.Fc or IL-17C.Fc at 100 ng/ml for 18 hours and theconditioned media were assayed for IL-6 as described in EXAMPLE 28. FIG.36 (part B.) shows the human leukemic cell line, THP-1, which wastreated with the same cytokines (100 ng/ml) as above under the sameconditions wherein the supernatants were assayed for the level of TNF-αrelease. Results are expressed as the mean+/−SE of triplicatedeterminations from one representative experiment.

FIG. 37 shows a time course representing the dependence of IL-17B(PRO1031) and IL-17C (PRO1122) activated TNF-α release from THP-1 cells.In FIG. 37 (part A.), THP-1 cells were incubated with 100 ng/ml (2.2 nM)of IL-17B.Fc or IL-17C.Fc for 0.5 to 32 hours, the conditioned mediaharvested, and the TNF-α concentration quantitated as described inEXAMPLE 28. In FIG. 37 (part B.), THP-1 cells were treated with theIL-17B.Fc and IL-17C.Fc at a concentration range from 0 to 120 nM for 18hours and the TNF-α release determined.

FIG. 38 shows FACS analysis of the binding of IL-17B.Fc and IL-17C.Fc toTHP-1 cells as described in EXAMPLE 29. THP-1 cells were incubated withIL-17B.Fc (FIG. 38 partA.) or IL-17C.Fc (FIG. 38 partB.) or control Fcfusion proteins in PBS (5% horse serum) and followed by addition of FITCconjugated anti-Fc secondary antibodies.

FIG. 39 shows the effect of IL-17 on articular cartilage. Cartilageexplants were cultured with the indicated concentration of IL-17 alone(solid) or in the presence of IL-1α at the indicated concentration(hatched) or IL-1ra (IL-1 receptor antagonist, R & D Systems, 1 μg/ml,for 72 hours). Release of proteoglycans (PG) into the media (top panel)indicates matrix breakdown. Matrix synthesis was determined byincorporation of ³⁵S-sulfate into the tissue (bottom panel).

FIG. 40 shows the effect of IL-17 on the release of nitric oxide.Explants were treated with IL-17 (10 ng/ml) alone (left columns) or inthe presence of IL-1α (10 ng/ml) (right columns). After 48 hours, mediawas assayed for nitrite concentration.

FIG. 41 shows the effect of nitric oxide (NO) on IL-17 induced changesin matrix metabolism. Explants were treated with IL-17 (5 ng/ml) alone(+) or with an irreversible inhibitor of nitric oxide synthase, NOS(L-NIO, Caymen Chemical, 0.5 mM). After 72 hours of treatment, media wasassayed for nitrite (FIG. 41 part A) and proteoglycans (Pgs) (FIG. 41part B). FIG. 41 part C shows proteoglycan synthesis as determined byincorporation of ³⁵S-sulphate into the tissue.

FIG. 42 shows the effect of the inhibition of nitric oxide (NO) on IL-17induced changes in proteoglycan (PG) metabolism. Articular cartilageexplants were treated with IL-1α (5 ng/ml) alone (+) or with inhibitorsof NOS (L-NIO or L-NIL) (NIL, reversible NOS inhibitor, Caymen Chemical)or IL-1ra (IL-1 receptor antagonist, R & D Systems, 1 μg/ml). After 72hours of treatment, media was assayed for nitrite concentration andamount of proteoglycans. Matrix synthesis was determined byincorporation of ³⁵S-sulphate into the tissue.

FIG. 43 shows the effect of IL-17C (PRO1122) on articular cartilage.Explants were treated with IL-17C at 1% or 0.1% in the absence (leftmost3 columns) or presence (rightmost 3 columns) of IL-1∀(+) (10 ng/ml.Proteoglycan (PG) release and synthesis are shown as amount abovecontrol.

FIG. 44 shows the relative expression of the human IL-17 family in themouse model of inflammatory bowel disease [IBD] as demonstrated by-delta Ct values relative to GAPDH. IL-17 shows enhanced expression inthis mouse model during mild and severe stages of inflammatory boweldisease. In contrast, IL-17E (PRO10272) demonstrates a marked decreasein expression during severe stages of IBD, whereas IL-17B (PRO1031)demonstrates a moderate decrease in expression in severe IBD.

FIG. 45 shows a time course study which measures the relative expressionof IL-17D (PRO21175) in a mouse model of stroke over the first 72 hours.IL-17D expression in the brain dramatically decreases from the timestroke is induced to the endpoint of 72 hours.

FIGS. 46A-46C shows the effect of IL-17E (PRO10272) on human articularcartilage. FIG. 46A. demonstrates inhibition of matrix synthesis. Humanarticular cartilage was treated with various concentrations of IL-17Eand matrix synthesis was determined by measuring incorporation of³⁵S-sulfate as described in EXAMPLE 30. FIG. 46B. demonstrates theeffect of IL-17E on inducing nitric oxide production. Human articularcartilage was treated with various concentrations of IL-17E and nitricoxide production was measured as described in EXAMPLE 30. FIG. 46C.demonstrates the effect of IL-17E on inducing IL-6 production in humanarticular cartilage. Human articular cartilage was treated with variousconcentrations of IL-17E, and production of IL-6 was measured by anELISA assay as described in EXAMPLE 28.

FIGS. 47A-47C show production of IL-17F (PRO20110) by activated T cellsand stimulation of cytokine production. FIG. 47A demonstrates theexpression of IL-17F in T cells. Relative mRNA expression is shown. PI:treated with PMA and inomycin. FIG. 47B shows induction of IL-8 infibroblasts by IL-17F. FIG. 47C shows induction of G-CSF in fibroblastsby IL-17F. Human primary foreskin fibroblasts were cultured for 24 hoursin the presence of the indicated concentrations of IL-17F. Conditionedmedium was then analyzed by ELISA for the presence of IL-8 and G-CSF.

FIGS. 48A-48F show the effect of IL-17F (PRO20110) and IL-17 on porcineand human cartilage. Porcine articular cartilage explants (FIGS.48A-48C) were treated with 0.06 nM IL-1α, or varying concentrations(0.1, 1 or 10 nM) of IL-17F or IL-17. FIG. 48A shows the effect ofIL-17F and IL-17 on proteoglycan breakdown, FIG. 48B shows the effect ofIL-17F and IL-17 on proteoglycan synthesis, and FIG. 48C shows theeffect of IL-17F and IL-17 on IL-6 production, respectively. Datarepresents the average of five independent samples +/−SEM. Humanarticular cartilage (from 65 year old caucasian female) explants (FIGS.48D-48F) were treated with 0.06 nM IL-1α, or varying concentrations(0.1, 1 or 10 nM) of IL-17F or IL-17. FIG. 48D shows the effect ofIL-17F and IL-17 on proteoglycan breakdown, FIG. 48E shows the effect ofIL-17F and IL-17 on proteoglycan synthesis, and FIG. 48F shows theeffect of IL-17F and IL-17 on IL-6 production, respectively. Datarepresents the average of five independent samples+/−SEM.

FIG. 49 shows the structure of IL-17F. FIG. 49A shows a ribbon trace ofthe IL-17F monomer. β-strands are labeled. Disulfides are shown asball-and-stick representation with the sulfur atoms colored yellow.Approximate positions of the additional cysteines are shown as orangeballs. Inset shows a cartoon representation of the canonical knot. FIG.49B shows the ribbon trace of the IL-17F dimer in red and blue.Disulfides are shown as in FIG. 49A. FIG. 49C shows the structure of NGFfrom the NGF-TrkA complex (Weismann et al. Nature 401:184-188 (1999)). Adisordered loop connects strands 2 and 3.

FIG. 50 shows the sequence alignment of IL-17F with other IL-17 familymembers. Regions of identity and conserved sequences between IL-17 andIL-17F are highlighted in green and yellow, respectively. When the otherfamily members also have conserved or identical residues in theseregions, they are similarly colored. Cysteine residues are indicated inorange. The conserved serines that replace the canonical knot cysteinesare highlighted with white letters. Disulfide bonds which are expectedto be conserved in all IL-17s are indicated by a black line connectingthe bonded cystines. The two cystines which form the inter-chaindisulfide in IL-17F are marked with an asterisk. Secondary structuralelements in IL-17F are shown above the sequences as blue arrows(f3-strands) or cylinders (a-helix). Residue numbering is from the startof the mature sequences.

FIGS. 51A-51C show a comparison of IL-17F (PRO20110) and IL-17 molecularstructure. Two orthogonal views, “side” (A) and “front” (B) of themolecular surface of IL-17F colored according to sequence conservationbetween IL-17 and IL-17F as shown in FIG. 50. The surface of residuesthat are identical between the two proteins are colored green,homologous residues are colored yellow, while residues that differsignificantly are colored white. The view in (B) is orientedapproximately 15E rotated from the view in FIG. 49B). Residues formingthe cavity are labeled. FIG. 51C is a “cut-away” view of the surface inFIG. 51B showing how the large cavities on either side of IL-17Fpenetrate deeply into the body of the dimer.

FIGS. 52A-52C shows a comparison of the IL-17F surface and the TrkAbinding site on NGF. FIGS. 52A and 52B show the molecular structure ofIL-17F is oriented as in FIG. 51. IL-17F is colored according to theelectrostatic surface potential: red, −5 kT, white, 0 kT; and blue, +5kT. The positions of the cavities are indicated by the circles. FIG. 52Cshows the molecular structure of NGF in the same orientation as IL-17Fin panel (B); domain 5 of TrkA is shown as a green ribbon (Weismann etal., Nature 401:184-188 (1999); pdb code 1WWW).

FIG. 53 depicts the predicted protein sequence of murine IL-17E. Thepartial signal sequence and mature protein sequence of mIL-17E (SEQ IDNO:41) is aligned with its human ortholog IL-17E (SEQ ID NO:6).

FIG. 54 shows the tissue distribution of IL-17E in human and mouse. ThemRNA expression of mIL-17E was examined by real time RT-PCR assays usingmIL-17E specific primers and probe. Relative expression is shown.

FIG. 55 demonstrates that mouse IL-17E transgenics are growth retardedin both females and males as compared to wild-type mice (body weightmeasurements were made from 1 week to 6 weeks of age).

FIG. 56 shows the gross appearance of a mIL-17E TG mouse (TG shown onthe right) compared to a non-TG littermate (WT shown on the left). Thetransgenic mouse (left) is smaller than the non-TG mouse (right). ThemIL-17E transgenic has visibly jaundice skin (yellow-tinged).

FIG. 57 shows mouse IL-17E transgenics (TG) have elevated totalbilirubin and liver enzymes compared with non-transgenic (Non-TG) enzymepanels.

FIGS. 58A-58E shows mRNA levels of various genes in the TG versus non-TGmice. Gene expression in various tissues was examined by real-timequantitative RT-PCR using gene-specific primers and probes as describedin EXAMPLE 35. Relative mRNA expression values (TG versus non-TG) areshown as fold increase. FIG. 58A shows the gene expression profile inthe liver. FIG. 58B (right panel) shows the gene expression profile inthe kidney; FIG. 58C shows the gene expression profile in lung tissues.FIG. 58D shows the gene expression profile in the spleen; FIG. 58E showsthe gene expression profile in the intestines.

FIG. 59 demonstrates the up-regulation of IL-17E receptor (mIL-17ER)expression in mouse IL-17E transgenics versus non-transgenics asdetermined by Taqman analysis in lung, kidney, liver, spleen and hearttissue.

FIG. 60 shows the elevated serum levels of IL-5, IL-13 and TNF-α inmIL-17E transgenics.

FIG. 61 shows the elevated serum levels of IgE and IgG1, but not IgG2 inmIL-17E transgenics.

FIG. 62 shows FACS and hemotology analyses of PBMC from TG mice. FIG.62A shows a reduced CD3⁺ T cell and CD19⁺ B cell populations in PBMCfrom TG mice. Percentage for each cell population is shown. FIG. 62Bshows an increased GR-1⁺ neutrophil population in PBMC of TG mice.

FIG. 63 shows changes in absolute cell counts (×10⁶/ml) of circulatingneutrophils, eosinophils and lymphocytes in TG mice.

FIG. 64 demonstrates elevated G-CSF levels in mIL-17E transgenics ascompared with non-transgenic mice.

FIG. 65 shows that IL-17E directly stimulates G-CSF production fromstromal cells NIH3T3 and ST2. Far right panel shows confirmation byELISA of G-CSF protein stimulated production from NIH3T3 cells.

FIG. 66 shows the comparison of histological changes in mIL-17E TG mice(B and D) and non-TG littermates (A and C). Part A shows the portal areaof the liver from a wild-type littermates. Part B shows the liver from amIL-17E transgenic mouse demonstrating severe periportal inflammation,fibrosis and adenomatous hyperplasia of bile ducts. Arrow depictshypertrophied bile duct epithelial cells distended with eosinophilichomogenous or crystalline material. Part C shows lung from a wild-typelittermate with normal alveoli and brochioles. Part D shows lung from amIL-17E transgenic mouse with diffues inflammation within alveolarspaces, septa and around airways. Arrow demonstrates the hyperplasticbronchiole epithelium.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods. Theterm “PRO polypeptide” refers to each individual PRO/number polypeptidedisclosed herein. All disclosures in this specification which refer tothe “PRO polypeptide” refer to each of the polypeptides individually aswell as jointly. For example, descriptions of the preparation of,purification of, derivation of, formation of antibodies to or against,administration of, compositions containing, treatment of a disease with,etc., pertain to each polypeptide of the invention individually. Theterm “PRO polypeptide” also includes variants of the PRO/numberpolypeptides disclosed herein.

A “native sequence PRO polypeptide” comprises a polypeptide having thesame amino acid sequence as the corresponding PRO polypeptide derivedfrom nature. Such native sequence PRO polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence PRO polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of the specific PROpolypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

The PRO polypeptide “extracellular domain” or “ECD” refers to a form ofthe PRO polypeptide which is essentially free of the transmembrane andcytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have lessthan 1% of such transmembrane and/or cytoplasmic domains and preferably,will have less than 0.5% of such domains. It will be understood that anytransmembrane domains identified for the PRO polypeptides of the presentinvention are identified pursuant to criteria routinely employed in theart for identifying that type of hydrophobic domain. The exactboundaries of a transmembrane domain may vary but most likely by no morethan about 5 amino acids at either end of the domain as initiallyidentified herein. Optionally, therefore, an extracellular domain of aPRO polypeptide may contain from about 5 or fewer amino acids on eitherside of the transmembrane domain/extracellular domain boundary asidentified in the Examples or specification and such polypeptides, withor without the associated signal peptide, and nucleic acid encodingthem, are contemplated by the present invention.

The approximate location of the “signal peptides” of the various PROpolypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that t_(y)pe ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng., 10:1-6(1997) and von Heinje et al., Nucl. Acids. Res., 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“PRO polypeptide variant” means an active PRO polypeptide as definedabove or below having at least about 80% amino acid sequence identitywith a full-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the—or C-terminusof the full-length native amino acid sequence. Ordinarily, a PROpolypeptide variant will have at least about 80% amino acid sequenceidentity, alternatively at least about 81% amino acid sequence identity,alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to afull-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length PRO polypeptide sequenceas disclosed herein. Ordinarily, PRO variant polypeptides are at leastabout 10 amino acids in length, alternatively at least about 20 aminoacids in length, alternatively at least about 30 amino acids in length,alternatively at least about 40 amino acids in length, alternatively atleast about 50 amino acids in length, alternatively at least about 60amino acids in length, alternatively at least about 70 amino acids inlength, alternatively at least about 80 amino acids in length,alternatively at least about 90 amino acids in length, alternatively atleast about 100 amino acids in length, alternatively at least about 150amino acids in length, alternatively at least about 200 amino acids inlength, alternatively at least about 300 amino acids in length, or more.

“Percent (%) amino acid sequence identity” with respect to the PROpolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific PRO polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of the PROpolypeptide of interest having a sequence derived from the native PROpolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the PRO polypeptide of interest is beingcompared which may be a PRO variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest. For example, in the statement “a polypeptidecomprising an the amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of interest andthe amino acid sequence B is the amino acid sequence of the PROpolypeptide of interest.

Percent amino acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be downloaded from the National Center for Biotechnology Information(NCBI) website or otherwise obtained from the National Institute ofHealth, Bethesda, Md. NCBI-BLAST2 uses several search parameters,wherein all of those search parameters are set to default valuesincluding, for example, unmask=yes, strand=all, expected occurrences=10,minimum low complexity length=15/5, multi-pass e-value=0.01, constantfor multi-pass=25, dropoff for final gapped alignment=25 and scoringmatrix=BLOSUM62

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

“PRO variant polynucleotide” or “PRO variant nucleic acid sequence”means a nucleic acid molecule which encodes an active PRO polypeptide asdefined below and which has at least about 80% nucleic acid sequenceidentity with a nucleotide acid sequence encoding a full-length nativesequence PRO polypeptide sequence as disclosed herein, a full-lengthnative sequence PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Ordinarily,a PRO variant polynucleotide will have at least about 80% nucleic acidsequence identity, alternatively at least about 81% nucleic acidsequence identity, alternatively at least about 82% nucleic acidsequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity with a nucleic acid sequence encoding a full-lengthnative sequence PRO polypeptide sequence as disclosed herein, afull-length native sequence PRO polypeptide sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal sequence, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Variants do not encompass the native nucleotidesequence.

Ordinarily, PRO variant polynucleotides are at least about 30nucleotides in length, alternatively at least about 60 nucleotides inlength, alternatively at least about 90 nucleotides in length,alternatively at least about 120 nucleotides in length, alternatively atleast about 150 nucleotides in length, alternatively at least about 180nucleotides in length, alternatively at least about 210 nucleotides inlength, alternatively at least about 240 nucleotides in length,alternatively at least about 270 nucleotides in length, alternatively atleast about 300 nucleotides in length, alternatively at least about 450nucleotides in length, alternatively at least about 600 nucleotides inlength, alternatively at least about 900 nucleotides in length, or more.

“Percent (%) nucleic acid sequence identity” with respect toPRO-encoding nucleic acid sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the PRO nucleic acid sequence of interest, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

Percent nucleic acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be downloaded from the National Center for Biotechnology Information(NCBI) website or otherwise obtained from the National Institute ofHealth, Bethesda, Md. NCBI-BLAST2 uses several search parameters,wherein all of those search parameters are set to default valuesincluding, for example, unmask=yes, strand=all, expected occurrences=10,minimum low complexity length=15/5, multi-pass e-value=0.01, constantfor multi-pass=25, dropoff for final gapped alignment=25 and scoringmatrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In other embodiments, PRO variant polynucleotides are nucleic acidmolecules that encode an active PRO polypeptide and which are capable ofhybridizing, preferably under stringent hybridization and washconditions, to nucleotide sequences encoding a full-length PROpolypeptide as disclosed herein. PRO variant polypeptides may be thosethat are encoded by a PRO variant polynucleotide.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” PRO polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-PRO monoclonal antibodies (includingagonist, antagonist, and neutralizing antibodies), anti-PRO antibodycompositions with polyepitopic specificity, single chain anti-PROantibodies, and fragments of anti-PRO antibodies (see below). The term“monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and %SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of native PROpolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of a PROpolypeptide may comprise contacting a PRO polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the PROpolypeptide.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.,8(10):1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH 1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab' in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a PRO polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

The term “modulate” means to affect (e.g., either upregulate,downregulate or otherwise control) the level of a signaling pathway.Cellular processes under the control of signal transduction include, butare not limited to, transcription of specific genes, normal cellularfunctions, such as metabolism, proliferation, differentiation, adhesion,apoptosis and survival, as well as abnormal processes, such astransformation, blocking of differentiation and metastasis.

“Active” or “activity” for the purposes herein refers to form(s) of aPRO polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO polypeptides, wherein“biological” activity refers to a biological function (either inhibitoryor stimulatory) caused by a native or naturally-occurring PROpolypeptide other than the ability to induce the production of anantibody against an antigenic epitope possessed by a native ornaturally-occurring PRO polypeptide and an “immunological” activityrefers to the ability to induce the production of an antibody against anantigenic epitope possessed by a native or naturally-occurring PROpolypeptide. One preferred biological activity includes inducingactivation of NF-κB and stimulation of the production of theproinflammatory chemokine IL-8. Another preferred biological activityincludes stimulation of peripheral blood mononuclear cells or CD4⁺cells. Another preferred biological activity includes stimulation of theproliferation of T-lymphocytes. Another preferred biological activityincludes, for example, the release of TNF-α from THP 1 cells. Analternative activity is the reduction in IL-1a induced NO (nitric oxide)production from articular cartilage. Another activity includes anenhancement of matrix synthesis in articular cartilage. Alternatively,another activity includes promoting breakdown of articular cartilagematrix as well as inhibiting matrix synthesis. Another preferredbiological activity includes modulating the level of the interleukin-17signalling pathway during mild to severe stages of inflammatory boweldisease or during stroke.

An “immunological” activity refers only to the ability to induce theproduction of an antibody against an antigenic epitope possessed by anative or naturally-occurring PRO polypeptide.

“Degenerative cartilagenous disorder” describes a host of disorders thatis characterized principally by the destruction of the cartilage matrix.Additional pathologies includes nitric oxide production, and elevatedproteoglycan breakdown. Exemplary disorders encompassed within thisdefinition, include, for example, arthritis (e.g., osteoarthritis,rheumatoid arthritis, psoriatic arthritis).

The term “immune related disease” means a disease in which a componentof the immune system of a mammal causes, mediates or otherwisecontributes to a morbidity in the mammal. Also included are diseases inwhich stimulation or intervention of the immune response has anameliorative effect on progression of the disease. Included within thisterm are immune-mediated inflammatory diseases, non-immune-mediatedinflammatory diseases, infectious diseases, immunodeficiency diseases,neoplasia, etc.

The term “T cell mediated disease” means a disease in which T cellsdirectly or indirectly mediate or otherwise contribute to a morbidity ina mammal. The T cell mediated disease may be associated with cellmediated effects, lymphokine mediated effects, etc., and even effectsassociated with B cells if the B cells are stimulated, for example, bythe lymphokines secreted by T cells.

Examples of immune-related and inflammatory diseases, some of which areimmune or T cell mediated, which can be treated according to theinvention include systemic lupus erythematosis, rheumatoid arthritis,juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis(scleroderma), idiopathic inflammatory myopathies (dermatomyositis,polymyositis), Sjögren's syndrome, systemic vasculitis, sarcoidosis,autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy, hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis, inflammatory bowel disease(ulcerative colitis: Crohn's disease), gluten-sensitive enteropathy, andWhipple's disease, autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis, allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria, immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus-host-disease. Infectiousdiseases including viral diseases such as AIDS (HIV infection),hepatitis A, B, C, D, and E, herpes, etc., bacterial infections, fungalinfections, protozoal infections and parasitic infections. The term“effective amount” is a concentration or amount of a PRO polypeptideand/or agonist/antagonist which results in achieving a particular statedpurpose. An “effective amount” of a PRO polypeptide or agonist orantagonist thereof may be determined empirically. Furthermore, a“therapeutically effective amount” is a concentration or amount of a PROpolypeptide and/or agonist/antagonist which is effective for achieving astated therapeutic effect. This amount may also be determinedempirically.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel (Taxotere, Rhône-Poulenc Rorer, Antony,France), toxotere, methotrexate, cisplatin, melphalan, vinblastine,bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone,vincristine, vinorelbine, carboplatin, teniposide, daunomycin,carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (seeU.S. Pat. No. 4,675,187), melphalan and other related nitrogen mustards.Also included in this definition are hormonal agents that act toregulate or inhibit hormone action on tumors such as tamoxifen andonapristone.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially cancer celloverexpressing any of the genes identified herein, either in vitro or invivo. Thus, the growth inhibitory agent is one which significantlyreduces the percentage of cells overexpressing such genes in S phase.Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce G1 arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxol, and topo II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al., (W BSaunders: Philadelphia, 1995), especially p. 13.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, or IL-17; a tumor necrosis factorsuch as TNF-α or TNF-β; and other polypeptide factors including leukemiainhibitory factor (LIF) and kit ligand (KL). As used herein, the termcytokine includes proteins from natural sources or from recombinant cellculture and biologically active equivalents of the native sequencecytokines.

TABLE 1 /*  *  * C-C increased from 12 to 15  * Z is average of EQ  * Bis average of ND  * match with stop is _M; stop−stop = 0; J (joker)match = 0  */ #define _M −8 /* value of a match with a stop */ int_day[26][26] = { /* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/ /* A */ { 2, 0,−2, 0, 0,−4, 1,−1,−1, 0,−1,−2,−1, 0,_M, 1, 0,−2, 1, 1,0, 0,−6, 0,−3, 0}, /* B */ { 0, 3,−4, 3, 2,−5, 0, 1,−2, 0, 0,−3,−2,2,_M,−1, 1, 0, 0, 0, 0,−2,−5, 0,−3, 1}, /* C */{−2,−4,15,−5,−5,−4,−3,−3,−2, 0,−5,−6,−5,−4,_M,−3,−5,−4, 0,−2, 0,−2,−8,0, 0,−5}, /* D */ { 0, 3,−5, 4, 3,−6, 1, 1,−2, 0, 0,−4,−3, 2,_M,−1,2,−1, 0, 0, 0,−2,−7, 0,−4, 2}, /* E */ { 0, 2,−5, 3, 4,−5, 0, 1,−2, 0,0,−3,−2, 1,_M,−1, 2,−1, 0, 0, 0,−2,−7, 0,−4, 3}, /* F */{−4,−5,−4,−6,−5, 9,−5,−2, 1, 0,−5, 2, 0,−4,_M,−5,−5,−4,−3,−3, 0,−1, 0,0, 7,−5}, /* G */ { 1, 0,−3, 1, 0,−5, 5,−2,−3, 0,−2,−4,−3,0,_M,−1,−1,−3, 1, 0, 0,−1,−7, 0,−5, 0}, /* H */ {−1, 1,−3, 1, 1,−2,−2,6,−2, 0, 0,−2,−2, 2,_M, 0, 3, 2,−1,−1, 0,−2,−3, 0, 0, 2}, /* I */{−1,−2,−2,−2,−2, 1,−3,−2, 5, 0,−2, 2, 2,−2,_M,−2,−2,−2,−1, 0, 0, 4,−5,0,−1,−2}, /* J */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0}, /* K */ {−1, 0,−5, 0, 0,−5,−2, 0,−2, 0,5,−3, 0, 1,_M,−1, 1, 3, 0, 0, 0,−2,−3, 0,−4, 0}, /* L */{−2,−3,−6,−4,−3, 2,−4,−2, 2, 0,−3, 6, 4,−3,_M,−3,−2,−3,−3,−1, 0, 2,−2,0,−1,−2}, /* M */ {−1,−2,−5,−3,−2, 0,−3,−2, 2, 0, 0, 4, 6,−2,_M,−2,−1,0,−2,−1, 0, 2,−4, 0,−2,−1}, /* N */ { 0, 2,−4, 2, 1,−4, 0, 2,−2, 0,1,−3,−2, 2,_M,−1, 1, 0, 1, 0, 0,−2,−4, 0,−2, 1}, /* O */{_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M, 0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M}, /* P */ { 1,−1,−3,−1,−1,−5,−1, 0,−2,0,−1,−3,−2,−1,_M, 6, 0, 0, 1, 0, 0,−1,−6, 0,−5, 0}, /* Q */ { 0, 1,−5,2, 2,−5,−1, 3,−2, 0, 1,−2,−1, 1,_M, 0, 4, 1,−1,−1, 0,−2,−5, 0,−4, 3}, /*R */ {−2, 0,−4,−1,−1,−4,−3, 2,−2, 0, 3,−3, 0, 0,_M, 0, 1, 6, 0,−1, 0,−2,2, 0,−4, 0}, /* S */ { 1, 0, 0, 0, 0,−3, 1,−1,−1, 0, 0,−3,−2, 1,_M,1,−1, 0, 2, 1, 0,−1,−2, 0,−3, 0}, /* T */ { 1, 0,−2, 0, 0,−3, 0,−1, 0,0, 0,−1,−1, 0,_M, 0,−1,−1, 1, 3, 0, 0,−5, 0,−3, 0}, /* U */ { 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /*V */ { 0,−2,−2,−2,−2,−1,−1,−2, 4, 0,−2, 2, 2,−2,_M,−1,−2,−2,−1, 0, 0,4,−6, 0,−2,−2}, /* W */ {−6,−5,−8,−7,−7, 0,−7,−3,−5,0,−3,−2,−4,−4,_M,−6,−5, 2,−2,−5, 0,−6,17, 0, 0,−6}, /* X */ { 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /*Y */ {−3,−3, 0,−4,−4, 7,−5, 0,−1, 0,−4,−1,−2,−2,_M,−5,−4,−4,−3,−3, 0,−2,0, 0,10,−4}, /* Z */ { 0, 1,−5, 2, 3,−5, 0, 2,−2, 0, 0,−2,−1, 1,_M, 0,3, 0, 0, 0, 0,−2,−6, 0,−4, 4} }; /*  */ #include <stdio.h> #include<ctype.h> #define MAXJMP 16 /* max jumps in a diag */ #define MAXGAP 24/* don't continue to penalize gaps larger than this */ #define JMPS 1024/* max jmps in an path */ #define MX 4 /* save if there's at least MX−1bases since last jmp */ #define DMAT 3 /* value of matching bases */#define DMIS 0 /* penalty for mismatched bases */ #define DINS0 8 /*penalty for a gap */ #define DINS1 1 /* penalty per base */ #definePINS0 8 /* penalty for a gap */ #define PINS1 4 /* penalty per residue*/ struct jmp { short n[MAXJMP]; /* size of jmp (neg for dely) */unsigned short x[MAXJMP]; /* base no. of jmp in seq x */ }; /* limitsseq to 2{circumflex over ( )}16 −1 */ struct diag { int score; /* scoreat last jmp */ long offset; /* offset of prev block */ short ijmp; /*current jmp index */ struct jmp jp; /* list of jmps */ }; struct path {int spc; /* number of leading spaces */ short n[JMPS];/* size of jmp(gap) */ int x[JMPS];/* loc of jmp (last elem before gap) */ }; char*ofile; /* output file name */ char *namex[2]; /* seq names: getseqs( )*/ char *prog; /* prog name for err msgs */ char *seqx[2]; /* seqs:getseqs( ) */ int dmax; /* best diag: nw( ) */ int dmax0; /* final diag*/ int dna; /* set if dna: main( ) */ int endgaps; /* set if penalizingend gaps */ int gapx, gapy; /* total gaps in seqs */ int len0, len1; /*seq lens */ int ngapx, ngapy; /* total size of gaps */ int smax; /* maxscore: nw( ) */ int *xbm; /* bitmap for matching */ long offset; /*current offset in jmp file */ struct diag *dx; /* holds diagonals */struct path pp[2]; /* holds path for seqs */ char *calloc( ), *malloc(), *index( ), *strcpy( ); char *getseq( ), *g_calloc( ); /*Needleman-Wunsch alignment program  *  * usage: progs file1 file2 * where file1 and file2 are two dna or two protein sequences.  * Thesequences can be in upper- or lower-case an may contain ambiguity  * Anylines beginning with ‘;’, ‘>’ or ‘<’ are ignored  * Max file length is65535 (limited by unsigned short x in the jmp struct)  * A sequence with⅓ or more of its elements ACGTU is assumed to be DNA  * Output is in thefile “align.out”  *  * The program may create a tmp file in /tmp to holdinfo about traceback.  * Original version developed under BSD 4.3 on avax 8650  */ #include “nw.h” #include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1<<(‘D’−‘A’))|(1<<(‘N’−‘A’)), 4, 8, 16, 32, 64,128, 256, 0xFFFFFFF, 1<<10, 1<<11, 1<<12, 1<<13, 1<<14, 1<<15, 1<<16,1<<17, 1<<18, 1<<19, 1<<20, 1<<21, 1<<22, 1<<23, 1<<24,1<<25|(1<<(‘E’−‘A’))|(1<<(‘Q’−‘A’)) }; main(ac, av) main int ac; char*av[ ]; { prog = av[0]; if (ac != 3) { fprintf(stderr,“usage: %s file1file2\n”, prog); fprintf(stderr,“where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr,“The sequences can be inupper- or lower-case\n”); fprintf(stderr,“Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr,“Output is in the file\“align.out\”\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw( ); /* fill in the matrix, getthe possible jmps */ readjmps( ); /* get the actual jmps */ print( ); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ } /* dothe alignment, return best score: main( )  * dna: values in Fitch andSmith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw( ) nw { char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /*keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row0, row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index*/ register *col0, *col1; /* score for curr,last row */ register xx, yy; /* index into seqs */ dx = (struct diag*)g_calloc(“to get diags”, len0+len1+1, sizeof(struct diag)); ndely =(int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int*)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PINS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1 ; yy <= len1 ; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy = 1; yy <= len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0] − ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx =0; } ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis += (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps ∥ ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >= dely[yy]){ dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else { dely[yy] −=ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >= dely[yy]) {dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else ndely[yy]++; }/* update penalty for del in y seq;  * favor new del over ongong del  */if (endgaps ∥ ndelx < MAXGAP) { if (col1[yy−1] − ins0 >= delx) { delx =col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −= ins1; ndelx++; } }else { if (col1[yy−1] − (ins0+ins1) >= delx) { delx = col1[yy−1] −(ins0+ins1); ndelx = 1; } else ndelx++; } /* pick the maximum score;we're favoring  * mis over any del and delx over dely  */ ...nw id = xx− yy + len1 − 1; if (mis >= delx && mis >= dely[yy]) col1[yy] = mis;else if (delx >= dely[yy]) { col1[yy] = delx; ij = dx[id].ijmp; if(dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP && xx > dx[id].jp.x[ij]+MX)∥ mis > dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] = ndelx;dx[id].jp.x[ij] =xx; dx[id].score = delx; } else { col1[yy] = dely[yy];ij = dx[id].ijmp; if (dx[id].jp.n[0] && (!dna ∥ (ndely[yy] >= MAXJMP &&xx > dx[id].jp.x[ij]+MX) ∥ mis > dx[id].score+DINS0)) { dx[id].ijmp++;if (++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset= offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = −ndely[yy]; dx[id].jp.x[ij] = xx; dx[id].score =dely[yy]; } if (xx == len0 && yy < len1) { /* last col  */ if (endgaps)col1[yy] −= ins0+ins1*(len1−yy); if (col1[yy] > smax) { smax = col1[yy];dmax = id; } } } if (endgaps && xx < len0) col1[yy−1] −=ins0+ins1*(len0−xx); if (col1[yy−1] > smax) { smax = col1[yy−1]; dmax =id; } tmp = col0; col0 = col1; col1 = tmp; } (void) free((char *)ndely);(void) free((char *)dely); (void) free((char *)col0); (void) free((char*)col1); } /*  *  * print( ) -- only routine visible outside this module *  * static:  * getmat( ) -- trace back best path, count matches:print( )  * pr_align( ) -- print alignment of described in array p[ ]:print( )  * dumpblock( ) -- dump a block of lines with numbers, stars:pr_align( )  * nums( ) -- put out a number line: dumpblock( )  *putline( ) -- put out a line (name, [num], seq, [num]): dumpblock( )  *stars( ) - -put a line of stars: dumpblock( )  * stripname( ) -- stripany path and prefix from a seqname  */ #include “nw.h” #define SPC 3#define P_LINE 256 /* maximum output line */ #define P_SPC 3 /* spacebetween name or num and seq */ extern _day[26][26]; int olen; /* setoutput line length */ FILE *fx; /* output file */ print( ) print { intlx, ly, firstgap, lastgap; /* overlap */ if ((fx = fopen(ofile, “w”)) ==0) { fprintf(stderr,“%s: can't write %s\n”, prog, ofile); cleanup(1); }fprintf(fx, “<first sequence: %s (length = %d)\n”, namex[0], len0);fprintf(fx, “<second sequence: %s (length = %d)\n”, namex[1], len1);olen = 60; lx = len0; ly = len1; firstgap = lastgap = 0; if (dmax < len1− 1) { /* leading gap in x */ pp[0].spc = firstgap = len1 − dmax − 1; ly−= pp[0].spc; } else if (dmax > len1 − 1) { /* leading gap in y */pp[1].spc = firstgap = dmax − (len1 − 1); lx −= pp[1].spc; } if (dmax0 <len0 − 1) { /* trailing gap in x */ lastgap = len0 − dmax0 −1; lx −=lastgap; } else if (dmax0 > len0 − 1) { /* trailing gap in y */ lastgap= dmax0 − (len0 − 1); ly −= lastgap; } getmat(lx, ly, firstgap,lastgap); pr_align( ); } /*  * trace back the best path, count matches */ static getmat(lx, ly, firstgap, lastgap) getmat int lx, ly; /*“core” (minus endgaps) */ int firstgap, lastgap; /* leading trailingoverlap */ { int nm, i0, i1, siz0, siz1; char outx[32]; double pct;register n0, n1; register char *p0, *p1; /* get total matches, score  */i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] + pp[1].spc; p1 = seqx[1] +pp[0].spc; n0 = pp[1].spc + 1; n1 = pp[0].spc + 1; nm = 0; while ( *p0&& *p1 ) { if (siz0) { p1++; n1++; siz0−−; } else if (siz1) { p0++;n0++; siz1−−; } else { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if (n0++ ==pp[0].x[i0]) siz0 = pp[0].n[i0++]; if (n1++ == pp[1].x[i1]) siz1 =pp[1].n[i1++]; p0++; p1++; } } /* pct homology:  * if penalizingendgaps, base is the shorter seq  * else, knock off overhangs and takeshorter core  */ if (endgaps) lx = (len0 < len1)? len0 : len1; else lx =(lx < ly)? lx : ly; pct = 100.*(double)nm/(double)lx; fprintf(fx, “\n”);fprintf(fx, “<%d match%s in an overlap of %d: %.2f percentsimilarity\n”, nm, (nm == 1)? “” : “es”, lx, pct); fprintf(fx, “<gaps infirst sequence: %d”, gapx); ...getmat if (gapx) { (void) sprintf(outx, “(%d %s%s)”, ngapx, (dna)? “base”:“residue”, (ngapx == 1)? “”:“s”);fprintf(fx,“%s”, outx); fprintf(fx, “, gaps in second sequence: %d”,gapy); if (gapy) { (void) sprintf(outx, “ (%d %s%s)”, ngapy, (dna)?“base”:“residue”, (ngapy == 1)? “”:“s”); fprintf(fx,“%s”, outx); } if(dna) fprintf(fx, “\n<score: %d (match = %d, mismatch = %d, gap penalty= %d + %d per base)\n”, smax, DMAT, DMIS, DINS0, DINS1); elsefprintf(fx, “\n<score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %dper residue)\n”, smax, PINS0, PINS1); if (endgaps) fprintf(fx, “<endgapspenalized. left endgap: %d %s%s, right endgap: %d %s%s\n”, firstgap,(dna)? “base” : “residue”, (firstgap == 1)? “” : “s”, lastgap, (dna)?“base” : “residue”, (lastgap == 1)? “” : “s”); else fprintf(fx,“<endgaps not penalized\n”); }  static nm; /* matches in core -- forchecking */  static lmax; /* lengths of stripped file names */  staticij[2]; /* jmp index for a path */  static nc[2]; /* number at start ofcurrent line */  static ni[2]; /* current elem number -- for gapping */ static siz[2];  static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */  static charout[2][P_LINE]; /* output line */  static char star[P_LINE]; /* set bystars( ) */ /*  * print alignment of described in struct path pp[ ]  */static pr_align( )pr_align { int nn; /* char count */ int more; registeri; for (i = 0, lmax = 0; i < 2; i++) { nn = stripname(namex[i]); if(nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1; siz[i] = ij[i] = 0; ps[i] =seqx[i]; po[i] = out[i]; } for (nn = nm = 0, more = 1; more; ){...pr_align for (i = more = 0; i < 2; i++) { /*  * do we have more ofthis sequence?  */ if (!*ps[i]) continue; more++; if (pp[i].spc) { /*leading space */ *po[i]++ = ‘ ’; pp[i].spc−−; } else if (siz[i]) { /* ina gap */ *po[i]++ = ‘−’; siz[i]−−; } else { /* we're putting a seqelement  */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] = pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn ==olen ∥ !more && nn) { dumpblock( ); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align( )  */ static dumpblock( ) dumpblock { register i; for(i = 0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx);for (i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ ∥ *(po[i]) != ‘’)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars( ); putline(i);if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1) nums(i); } } } /* * put out a number line: dumpblock( )  */ static nums(ix) nums int ix;/* index in out[ ] holding seq line */ { char nline[P_LINE]; register i,j; register char *pn, *px, *py; for (pn = nline, i = 0; i < lmax+P_SPC;i++, pn++) *pn = ‘ ’; for (i = nc[ix], py = out[ix]; *py; py++, pn++) {if (*py == ‘ ’ ∥ *py == ‘−’) *pn = ‘ ’; else { if (i%10 == 0 ∥ (i == 1&& nc[ix] != 1)) { j = (i < 0)? −i : i; for (px = pn; j; j /= 10, px−−)*px = j%10 + ‘0’; if (i < 0) *px = ‘−’; } else *pn = ‘ ’; i++; } } *pn =‘\0’; nc[ix] = i; for (pn = nline; *pn; pn++) (void) putc(*pn, fx);(void) putc(‘\n’, fx); } /*  * put out a line (name, [num], seq, [num]):dumpblock( )  */ static putline(ix) putline int ix; { ...putline int i;register char *px; for (px = namex[ix], i = 0; *px && *px != ‘:’; px++,i++) (void) putc(*px, fx); for (; i < lmax+P_SPC; i++) (void) putc(‘ ’,fx); /* these count from 1:  * ni[ ] is current element (from 1)  * nc[] is number at start of current line  */ for (px = out[ix]; *px; px++)(void) putc(*px&0x7F, fx); (void) putc(‘\n’, fx); } /*  * put a line ofstars (seqs always in out[0], out[1]): dumpblock( )  */ static stars( )stars { int i; register char *p0, *p1, cx, *px; if (!*out[0] ∥ (*out[0]== ‘ ’ && *(po[0]) == ‘ ’) ∥  !*out[1] ∥ (*out[1] == ‘ ’ && *(po[1]) ==‘ ’)) return; px = star; for (i = lmax+P_SPC; i; i−−) *px++ = ‘ ’; for(p0 = out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0) &&isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm++; } elseif (!dna && _day[*p0−‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } elsecx = ‘ ’; *px++ = cx; } *px++ = ‘\n’; *px = ‘\0’; } /*  * strip path orprefix from pn, return len: pr_align( )  */ static stripname(pn)stripname char *pn; /* file name (may be path) */ { register char *px,*py; py = 0; for (px = pn; *px; px++) if (*px == ‘/’) py = px + 1; if(py) (void) strcpy(pn, py); return(strlen(pn)); } /*  * cleanup( ) --cleanup any tmp file  * getseq( ) -- read in seq, set dna, len, maxlen * g_calloc( ) -- calloc( ) with error checkin  * readjmps( ) -- get thegood jmps, from tmp file if necessary  * writejmps( ) -- write a filledarray of jmps to a tmp file: nw( )  */ #include “nw.h” #include<sys/file.h> char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */FILE *fj; int cleanup( ); /* cleanup tmp file */ long lseek( ); /*  *remove any tmp file if we blow  */ cleanup(i) cleanup int i; { if (fj)(void) unlink(jname); exit(i); } /*  * read, return ptr to seq, set dna,len, maxlen  * skip lines starting with ‘;’, ‘<’, or ‘>’  * seq in upperor lower case  */ char * getseq(file, len) getseq char *file; /* filename */ int *len; /* seq len */ { char line[1024], *pseq; register char*px, *py; int natgc, tlen; FILE *fp; if ((fp = fopen(file,“r”)) == 0) {fprintf(stderr,“%s: can't read %s\n”, prog, file); exit(1); } tlen =natgc = 0; while (fgets(line, 1024, fp)) { if (*line == ‘;’ ∥ *line ==‘<’ ∥ *line == ‘>’) continue; for (px = line; *px != ‘\n’; px++) if(isupper(*px) ∥ islower(*px)) tlen++; } if ((pseq =malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr,“%s: malloc( ) failedto get %d bytes for %s\n”, prog, tlen+6, file); exit(1); } pseq[0] =pseq[1] = pseq[2] = pseq[3] = ‘\0’; ...getseq py = pseq + 4; *len =tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line == ‘;’ ∥*line == ‘<’ ∥ *line == ‘>’) continue; for (px = line; *px != ‘\n’;px++) { if (isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ =toupper(*px); if (index(“ATGCU”,*(py−1))) natgc++; } } *py++ = ‘\0’; *py= ‘\0’; (void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); }char * g_calloc(msg, nx, sz) g_calloc char *msg; /* program, callingroutine */ int nx, sz; /* number and size of elements */ { char *px,*calloc( ); if ((px = calloc((unsigned)nx, (unsigned)sz)) == 0) { if(*msg) { fprintf(stderr, “%s: g_calloc( ) failed %s (n=%d, sz=%d)\n”,prog, msg, nx, sz); exit(1); } } return(px); } /*  * get final jmps fromdx[ ] or tmp file, set pp[ ], reset dmax: main( )  */ readjmps( )readjmps { int fd = −1; int siz, i0, i1; register i, j, xx; if (fj) {(void) fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) {fprintf(stderr, “%s: can't open( ) %s\n”, prog, jname); cleanup(1); } }for (i = i0 = i1 = 0, dmax0 = dmax, xx = len0; ; i++) { while (1) { for(j = dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ; ...readjmpsif (j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset,0); (void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void)read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));dx[dmax].ijmp = MAXJMP−1; } else break; } if (i >= JMPS) {fprintf(stderr, “%s: too many gaps in alignment\n”, prog); cleanup(1); }if (j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax +=siz; if (siz < 0) { /* gap in second seq */ pp[1].n[i1] = −siz; xx +=siz; /* id = xx − yy + len1 − 1  */ pp[1].x[i1] = xx − dmax + len1 − 1;gapy++; ngapy −= siz; /* ignore MAXGAP when doing endgaps */ siz = (−siz< MAXGAP ∥ endgaps)? −siz : MAXGAP; i1++; } else if (siz > 0) { /* gapin first seq */ pp[0].n[i0] = siz; pp[0].x[i0] = xx; gapx++; ngapx +=siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP ∥endgaps)? siz : MAXGAP; i0++; } } else break; } /* reverse the order ofjmps  */ for (j = 0, i0−−; j < i0; j++, i0−−) { i = pp[0].n[j];pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].x[j]; pp[0].x[j] =pp[0].x[i0]; pp[0].x[i0] = i; } for (j = 0, i1−−; j < i1; j++, i1−−) { i= pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j];pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = i; } if (fd >= 0) (void)close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /*  *write a filled jmp struct offset of the prev one (if any): nw( )  */writejmps(ix) writejmps int ix; { char *mktemp( ); if (!fj) { if(mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp( ) %s\n”, prog,jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 15 = 33.3%

TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 10 = 50%

TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acid sequenceidentity = (the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) divided by (thetotal number of nucleotides of the PRO-DNA nucleic acid sequence) = 6divided by 14 = 42.9%

TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) ComparisonNNNNLLLVV (Length = 9 nucleotides) DNA % nucleic acid sequence identity= (the number of identically matching nucleotides between the twonucleic acid sequences as determined by ALIGN-2) divided by (the totalnumber of nucleotides of the PRO-DNA nucleic acid sequence) = 4 dividedby 12 = 33.3%

II. Compositions and Methods of the Invention

A. Full-Length PRO Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO polypeptides. In particular, cDNAs encoding various PROpolypeptides have been identified and isolated, as disclosed in furtherdetail in the Examples below. It is noted that proteins produced inseparate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the full length native nucleic acidmolecules disclosed herein as well as all further native homologues andvariants included in the foregoing definition of PRO, will be referredto as “PRO/number”, regardless of their origin or mode of preparation.

As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO polypeptides and encoding nucleic acidsdescribed herein, Applicants have identified what is believed to be thereading frame best identifiable with the sequence information availableat the time.

B. PRO Polypeptide Variants

In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the native full-length sequence PRO or in various domainsof the PRO described herein, can be made, for example, using any of thetechniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the PRO that results in a change in the amino acidsequence of the PRO as compared with the native sequence PRO. Optionallythe variation is by substitution of at least one amino acid with anyother amino acid in one or more of the domains of the PRO. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the PRO with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

PRO polypeptide fragments are provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO polypeptide.

PRO fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, PRO polypeptide fragments share at leastone biological and/or immunological activity with the native PROpolypeptide disclosed herein.

In particular embodiments, conservative substitutions of interest areshown in Table 6 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of thePRO polypeptide are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

-   (1) hydrophobic: norleucine, met, ala, val, leu, ile;-   (2) neutral hydrophilic: cys, ser, thr;-   (3) acidic: asp, glu;-   (4) basic: asn, gln, his, lys, arg;-   (5) residues that influence chain orientation: gly, pro; and-   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis (Wells et al., Gene, 34:315 [1985]),restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 [1986]) or other known techniques can be performedon the cloned DNA to produce the PRO variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant (Cunningham and Wells,Science, 244: 1081-1085 [1989]). Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions (Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 [1976]). Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of PRO

Covalent modifications of PRO are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of a PRO polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC-terminal residues of the PRO. Derivatization with bifunctional agentsis useful, for instance, for crosslinking PRO to a water-insolublesupport matrix or surface for use in the method for purifying anti-PROantibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequencePRO. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO (for O-linkedglycosylation sites). The PRO amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the PRO polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of PRO comprises linking the PROpolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The PRO of the present invention may also be modified in a way to form achimeric molecule comprising PRO fused to another, heterologouspolypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of thePRO with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the PRO. The presence ofsuch epitope-tagged forms of the PRO can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe PRO to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the PRO with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

In yet a further embodiment, the PRO polypeptides of the presentinvention may also be modified in a way to form a chimeric moleculecomprising a PRO polypeptide fused to a leucine zipper. Various leucinezipper polypeptides have been described in the art. See, e.g.,Landschulz et al., Science 240:1759 (1988); WO 94/10308; Hoppe et al.,FEBS Letters, 344:1991 (1994); Maniatis et al., Nature 341:24 (1989). Itis believed that use of a leucine zipper fused to a PRO polypeptide maybe desirable to assist in dimerizing or trimerizing soluble PROpolypeptide in solution. Those skilled in the art will appreciate thatthe leucine zipper may be fused at either the—or C-terminal end of thePRO molecule.

D. Preparation of PRO

The description below relates primarily to production of PRO byculturing cells transformed or transfected with a vector containing PROnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare PRO. Forinstance, the PRO sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO may be chemically synthesized separately and combined using chemicalor enzymatic methods to produce the full-length PRO.

1. Isolation of DNA Encoding PRO

DNA encoding PRO may be obtained from a cDNA library prepared fromtissue believed to possess the PRO mRNA and to express it at adetectable level. Accordingly, human PRO DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

Libraries can be screened with probes (such as antibodies to the PRO oroligonucleotides of at least about 20-80 bases) designed to identify thegene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding PRO is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimizedThe oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The culture conditions, such as media, temperature, pH and the like, canbe selected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coll. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for PRO-encodingvectors. Saccharomyces cerevisiae is a commonly used lower eukaryotichost microorganism. Others include Schizosaccharomyces pombe [Beach andNurse, Nature, 290: 140 (1981); EP 139,383 published 2 May 1985];Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 [1991]) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 [1990]), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene,26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA,81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO are derivedfrom multicellular organisms. Examples of invertebrate cells includeinsect cells such as Drosophila S2 and Spodoptera Sf9 or Spodoptera High5 cells, as well as plant cells. Examples of useful mammalian host celllines include Chinese hamster ovary (CHO) and COS cells. More specificexamples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77:4216 [1980]); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23:243-251 [1980]); human lung cells (W138, ATCCCCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor(MMT 060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The PRO may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe PRO-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, 1pp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces ∀-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), orthe signal described in WO 90/13646 published 15 Nov. 1990. In mammaliancell expression, mammalian signal sequences may be used to directsecretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2u plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thePRO-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trpl gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the PRO by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,a-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 [1980]), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of PRO may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g., Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify PRO from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular PRO produced.

E. Uses for PRO

Nucleotide sequences (or their complement) encoding PRO have variousapplications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. PRO nucleic acid will also beuseful for the preparation of PRO polypeptides by the recombinanttechniques described herein.

The full-length native sequence PRO gene, or portions thereof, may beused as hybridization probes for a cDNA library to isolate thefull-length PRO cDNA or to isolate still other cDNAs (for instance,those encoding naturally-occurring variants of PRO or PRO from otherspecies) which have a desired sequence identity to the native PROsequence disclosed herein. Optionally, the length of the probes will beabout 20 to about 50 bases. The hybridization probes may be derived fromat least partially novel regions of the full length native nucleotidesequence wherein those regions may be determined without undueexperimentation or from genomic sequences including promoters, enhancerelements and introns of native sequence PRO. By way of example, ascreening method will comprise isolating the coding region of the PROgene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵5, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

Any EST sequences disclosed in the present application may similarly beemployed as probes, using the methods disclosed herein.

Other useful fragments of the PRO nucleic acids include antisense orsense oligonucleotides comprising a singe-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target PRO mRNA (sense) or PRODNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of the codingregion of PRO DNA. Such a fragment generally comprises at least about 14nucleotides, preferably from about 14 to 30 nucleotides. The ability toderive an antisense or a sense oligonucleotide, based upon a cDNAsequence encoding a given protein is described in, for example, Steinand Cohen (Cancer Res. 48:2659, [1988]) and van der Krol et al.(BioTechniques, 6:958, [1988]).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of PRO proteins.Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO 91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antisense or sense RNA or DNA molecules are generally at least about 5bases in length, about 10 bases in length, about 15 bases in length,about 20 bases in length, about 25 bases in length, about 30 bases inlength, about 35 bases in length, about 40 bases in length, about 45bases in length, about 50 bases in length, about 55 bases in length,about 60 bases in length, about 65 bases in length, about 70 bases inlength, about 75 bases in length, about 80 bases in length, about 85bases in length, about 90 bases in length, about 95 bases in length,about 100 bases in length, or more.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related PRO coding sequences.

Nucleotide sequences encoding a PRO can also be used to constructhybridization probes for mapping the gene which encodes that PRO and forthe genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for PRO encode a protein which binds toanother protein (example, where the PRO is a receptor), the PRO can beused in assays to identify the other proteins or molecules involved inthe binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor PRO can be used to isolate correlative ligand(s). Screeningassays can be designed to find lead compounds that mimic the biologicalactivity of a native PRO or a receptor for PRO. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds. The assays can be performed in a varietyof formats, including protein-protein binding assays, biochemicalscreening assays, immunoassays and cell based assays, which are wellcharacterized in the art.

Nucleic acids which encode PRO or its modified forms can also be used togenerate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding PRO can be used to clone genomic DNA encodingPRO in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding PRO. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for PRO transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding PRO introduced into the germ lineof the animal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding PRO. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PRO can be used to construct aPRO “knock out” animal which has a defective or altered gene encodingPRO as a result of homologous recombination between the endogenous geneencoding PRO and altered genomic DNA encoding PRO introduced into anembryonic stem cell of the animal. For example, cDNA encoding PRO can beused to clone genomic DNA encoding PRO in accordance with establishedtechniques. A portion of the genomic DNA encoding PRO can be deleted orreplaced with another gene, such as a gene encoding a selectable markerwhich can be used to monitor integration. Typically, several kilobasesof unaltered flanking DNA (both at the 5′ and 3′ ends) are included inthe vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for adescription of homologous recombination vectors]. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see, e.g., Li et al., Cell, 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras [see, e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of the PROpolypeptide.

Nucleic acid encoding the PRO polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA, 83:4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g., by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology, 11: 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g., capsid proteins or fragments thereof tropic fora particular cell t_(y)pe, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem., 262: 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA, 87: 3410-3414 (1990). For review of gene marking andgene therapy protocols see Anderson et al., Science, 256: 808-813(1992).

The PRO polypeptides described herein may also be employed as molecularweight markers for protein electrophoresis purposes and the isolatednucleic acid sequences may be used for recombinantly expressing thosemarkers.

The nucleic acid molecules encoding the PRO polypeptides or fragmentsthereof described herein are useful for chromosome identification. Inthis regard, there exists an ongoing need to identify new chromosomemarkers, since relatively few chromosome marking reagents, based uponactual sequence data are presently available. Each PRO nucleic acidmolecule of the present invention can be used as a chromosome marker.

The PRO polypeptides and nucleic acid molecules of the present inventionmay also be used diagnostically for tissue typing, wherein the PROpolypeptides of the present invention may be differentially expressed inone tissue as compared to another, preferably in a diseased tissue ascompared to a normal tissue of the same tissue type. PRO nucleic acidmolecules will find use for generating probes for PCR, Northernanalysis, Southern analysis and Western analysis.

The PRO polypeptides described herein may also be employed astherapeutic agents. The PRO polypeptides of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the PRO product hereof is combined in admixturewith a pharmaceutically acceptable carrier vehicle. Therapeuticformulations are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.,injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

When in vivo administration of a PRO polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 μg/kg/day to 10 mg/kg/day, depending upon the routeof administration.

Guidance as to particular dosages and methods of delivery is provided inthe literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344;or 5,225,212. It is anticipated that different formulations will beeffective for different treatment compounds and different disorders,that administration targeting one organ or tissue, for example, maynecessitate delivery in a manner different from that to another organ ortissue.

Where sustained-release administration of a PRO polypeptide is desiredin a formulation with release characteristics suitable for the treatmentof any disease or disorder requiring administration of the PROpolypeptide, microencapsulation of the PRO polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology, 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

The sustained-release formulations of these proteins were developedusing poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1-41.

This invention encompasses methods of screening compounds to identifythose that mimic the PRO polypeptide (agonists) or prevent the effect ofthe PRO polypeptide (antagonists). Screening assays for antagonist drugcandidates are designed to identify compounds that bind or complex withthe PRO polypeptides encoded by the genes identified herein, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a PRO polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the PRO polypeptide encoded by the gene identified herein orthe drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the PRO polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for the PROpolypeptide to be immobilized can be used to anchor it to a solidsurface. The assay is performed by adding the non-immobilized component,which may be labeled by a detectable label, to the immobilizedcomponent, e.g., the coated surface containing the anchored component.When the reaction is complete, the non-reacted components are removed,e.g., by washing, and complexes anchored on the solid surface aredetected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular PRO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 [1989]);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 [1991]) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA,89:5789-5793 (1991). Many transcriptional activators, such as yeastGAL4, consist of two physically discrete modular domains, one acting asthe DNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for 3-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding a PROpolypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the PRO polypeptide may be added to a cellalong with the compound to be screened for a particular activity and theability of the compound to inhibit the activity of interest in thepresence of the PRO polypeptide indicates that the compound is anantagonist to the PRO polypeptide. Alternatively, antagonists may bedetected by combining the PRO polypeptide and a potential antagonistwith membrane-bound PRO polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. The PROpolypeptide can be labeled, such as by radioactivity, such that thenumber of PRO polypeptide molecules bound to the receptor can be used todetermine the effectiveness of the potential antagonist. The geneencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting.Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the PRO polypeptide and a cDNAlibrary created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to the PROpolypeptide. Transfected cells that are grown on glass slides areexposed to labeled PRO polypeptide. The PRO polypeptide can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase. Following fixation andincubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andre-transfected using an interactive sub-pooling and re-screeningprocess, eventually yielding a single clone that encodes the putativereceptor.

As an alternative approach for receptor identification, labeled PROpolypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeled PROpolypeptide in the presence of the candidate compound. The ability ofthe compound to enhance or block this interaction could then bemeasured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with PROpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of the PROpolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO polypeptide.

Another potential PRO polypeptide antagonist is an antisense RNA or DNAconstruct prepared using antisense technology, where, e.g., an antisenseRNA or DNA molecule acts to block directly the translation of mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241:456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the PRO polypeptide (antisense—Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of the PRO polypeptide.When antisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the PRO polypeptide, thereby blocking the normalbiological activity of the PRO polypeptide. Examples of small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules, preferably soluble peptides, and synthetic non-peptidylorganic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

Diagnostic and therapeutic uses of the herein disclosed molecules mayalso be based upon the positive functional assay hits disclosed anddescribed below

F. Tissue Distribution

The location of tissues expressing the PRO can be identified bydetermining mRNA expression in various human tissues. The location ofsuch genes provides information about which tissues are most likely tobe affected by the stimulating and inhibiting activities of the PROpolypeptides. The location of a gene in a specific tissue also providessample tissue for the activity blocking assays discussed below.

As noted before, gene expression in various tissues may be measured byconventional Southern blotting, Northern blotting to quantitate thetranscription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205[1980]), dot blotting (DNA analysis), or in situ hybridization, using anappropriately labeled probe, based on the sequences provided herein.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes.

Gene expression in various tissues, alternatively, may be measured byimmunological methods, such as immunohistochemical staining of tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.

Conveniently, the antibodies may be prepared against a native sequenceof a PRO polypeptide or against a synthetic peptide based on the DNAsequences encoding the PRO polypeptide or against an exogenous sequencefused to a DNA encoding a PRO polypeptide and encoding a specificantibody epitope. General techniques for generating antibodies, andspecial protocols for Northern blotting and in situ hybridization areprovided below.

G. Antibody Binding Studies

The activity of the PRO polypeptides can be further verified by antibodybinding studies, in which the ability of anti-PRO antibodies to inhibitthe effect of the PRO polypeptides, respectively, on tissue cells istested. Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies, the preparation of whichwill be described hereinbelow.

Antibody binding studies may be carried out in any known assay method,such as competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual ofTechniques, pp.147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of target protein in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies preferably are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme. For immunohistochemistry, the tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin, for example.

H. Cell-Based Assays

Cell-based assays and animal models for immune related diseases can beused to further understand the relationship between the genes andpolypeptides identified herein and the development and pathogenesis ofimmune related disease.

In a different approach, cells of a cell type known to be involved in aparticular immune related disease are transfected with the cDNAsdescribed herein, and the ability of these cDNAs to stimulate or inhibitimmune function is analyzed. Suitable cells can be transfected with thedesired gene, and monitored for immune function activity. Suchtransfected cell lines can then be used to test the ability of poly- ormonoclonal antibodies or antibody compositions to inhibit or stimulateimmune function, for example to modulate T-cell proliferation orinflammatory cell infiltration. Cells transfected with the codingsequences of the genes identified herein can further be used to identifydrug candidates for the treatment of immune related diseases.

In addition, primary cultures derived from transgenic animals (asdescribed below) can be used in the cell-based assays herein, althoughstable cell lines are preferred. Techniques to derive continuous celllines from transgenic animals are well known in the art (see, e.g.,Small et al., Mol. Cell. Biol., 5: 642-648 [1985]).

One suitable cell based assay is the mixed lymphocyte reaction (MLR).Current Protocols in Immunology, unit 3.12; edited by J E Coligan, A MKruisbeek, D H Marglies, E M Shevach, W Strober, National Institutes ofHealth, Published by John Wiley & Sons, Inc. In this assay, the abilityof a test compound to stimulate or inhibit the proliferation ofactivated T cells is assayed. A suspension of responder T cells iscultured with allogeneic stimulator cells and the proliferation of Tcells is measured by uptake of tritiated thymidine. This assay is ageneral measure of T cell reactivity. Since the majority of T cellsrespond to and produce IL-2 upon activation, differences inresponsiveness in this assay in part reflect differences in IL-2production by the responding cells. The MLR results can be verified by astandard lymphokine (IL-2) detection assay. Current Protocols inImmunology, above, 3.15, 6.3.

A proliferative T cell response in an MLR assay may be due to directmitogenic properties of an assayed molecule or to external antigeninduced activation. Additional verification of the T cell stimulatoryactivity of the PRO polypeptides can be obtained by a costimulationassay. T cell activation requires an antigen specific signal mediatedthrough the T-cell receptor (TCR) and a costimulatory signal mediatedthrough a second ligand binding interaction, for example, the B7 (CD80,CD86)/CD28 binding interaction. CD28 crosslinking increases lymphokinesecretion by activated T cells. T cell activation has both negative andpositive controls through the binding of ligands which have a negativeor positive effect. CD28 and CTLA-4 are related glycoproteins in the Igsuperfamily which bind to B7. CD28 binding to B7 has a positivecostimulation effect of T cell activation; conversely, CTLA-4 binding toB7 has a negative T cell deactivating effect. Chambers, C. A. andAllison, J. P., Curr. Opin. Immunol., (1997) 9:396. Schwartz, R. H.,Cell (1992) 71:1065; Linsley, P. S. and Ledbetter, J. A., Annu. Rev.Immunol. (1993) 11:191; June, C. H. et al., Immunol. Today (1994)15:321; Jenkins, M. K., Immunity (1994) 1:405. In a costimulation assay,the PRO polypeptides are assayed for T cell costimulatory or inhibitoryactivity.

PRO polypeptides, as well as other compounds of the invention, which arestimulators (costimulators) of T cell proliferation and agonists, e.g.,agonist antibodies, thereto as determined by MLR and costimulationassays, for example, are useful in treating immune related diseasescharacterized by poor, suboptimal or inadequate immune function. Thesediseases are treated by stimulating the proliferation and activation ofT cells (and T cell mediated immunity) and enhancing the immune responsein a mammal through administration of a stimulatory compound, such asthe stimulating PRO polypeptides. The stimulating polypeptide may, forexample, be a PRO polypeptide or an agonist antibody thereof.

Direct use of a stimulating compound as in the invention has beenvalidated in experiments with 4-1BB glycoprotein, a member of the tumornecrosis factor receptor family, which binds to a ligand (4-1BBL)expressed on primed T cells and signals T cell activation and growth.Alderson, M. E. et al., J. Immunol. 24:2219 (1994).

The use of an agonist stimulating compound has also been validatedexperimentally. Activation of 4-1BB by treatment with an agonistanti-4-1BB antibody enhances eradication of tumors. Hellstrom, I. andHellstrom, K. E., Crit. Rev. Immunol., 18:1 (1998). Immunoadjuvanttherapy for treatment of tumors, described in more detail below, isanother example of the use of the stimulating compounds of theinvention. An immune stimulating or enhancing effect can also beachieved by antagonizing or blocking the activity of a PRO which hasbeen found to be inhibiting in the MLR assay. Negating the inhibitoryactivity of the compound produces a net stimulatory effect. Suitableantagonists/blocking compounds are antibodies or fragments thereof whichrecognize and bind to the inhibitory protein, thereby blocking theeffective interaction of the protein with its receptor and inhibitingsignaling through the receptor. This effect has been validated inexperiments using anti-CTLA-4 antibodies which enhance T cellproliferation, presumably by removal of the inhibitory signal caused byCTLA-4 binding. Walunas, T. L. et al., Immunity, 1:405 (1994).

Alternatively, an immune stimulating or enhancing effect can also beachieved by administration of a PRO polypeptide which has vascularpermeability enhancing properties. Enhanced vacuolar permeability wouldbe beneficial to disorders which can be attenuated by local infiltrationof immune cells (e.g., monocytes, eosinophils, PMNs) and inflammation.

On the other hand, PRO polypeptides, as well as other compounds of theinvention, which are direct inhibitors of T cellproliferation/activation, lymphokine secretion, and/or vascularpermeability can be directly used to suppress the immune response. Thesecompounds are useful to reduce the degree of the immune response and totreat immune related diseases characterized by a hyperactive,superoptimal, or autoimmune response. This use of the compounds of theinvention has been validated by the experiments described above in whichCTLA-4 binding to receptor B7 deactivates T cells. The direct inhibitorycompounds of the invention function in an analogous manner. The use ofcompound which suppress vascular permeability would be expected toreduce inflammation. Such uses would be beneficial in treatingconditions associated with excessive inflammation.

Alternatively, compounds, e.g., antibodies, which bind to stimulatingPRO polypeptides and block the stimulating effect of these moleculesproduce a net inhibitory effect and can be used to suppress the T cellmediated immune response by inhibiting T cell proliferation/activationand/or lymphokine secretion. Blocking the stimulating effect of thepolypeptides suppresses the immune response of the mammal. This use hasbeen validated in experiments using an anti-IL2 antibody. In theseexperiments, the antibody binds to IL2 and blocks binding of IL2 to itsreceptor thereby achieving a T cell inhibitory effect.

I. Animal Models

The results of the cell based in vitro assays can be further verifiedusing in vivo animal models and assays for T-cell function. A variety ofwell known animal models can be used to further understand the role ofthe genes identified herein in the development and pathogenesis ofimmune related disease, and to test the efficacy of candidatetherapeutic agents, including antibodies, and other antagonists of thenative polypeptides, including small molecule antagonists. The in vivonature of such models makes them predictive of responses in humanpatients. Animal models of immune related diseases include bothnon-recombinant and recombinant (transgenic) animals. Non-recombinantanimal models include, for example, rodent, e.g., murine models. Suchmodels can be generated by introducing cells into syngeneic mice usingstandard techniques, e.g., subcutaneous injection, tail vein injection,spleen implantation, intraperitoneal implantation, implantation underthe renal capsule, etc.

Graft-versus-host disease occurs when immunocompetent cells aretransplanted into immunosuppressed or tolerant patients. The donor cellsrecognize and respond to host antigens. The response can vary from lifethreatening severe inflammation to mild cases of diarrhea and weightloss. Graft-versus-host disease models provide a means of assessing Tcell reactivity against MHC antigens and minor transplant antigens. Asuitable procedure is described in detail in Current Protocols inImmunology, above, unit 4.3.

An animal model for skin allograft rejection is a means of testing theability of T cells to mediate in vivo tissue destruction and a measureof their role in transplant rejection. The most common and acceptedmodels use murine tail-skin grafts. Repeated experiments have shown thatskin allograft rejection is mediated by T cells, helper T cells andkiller-effector T cells, and not antibodies. Auchincloss, H. Jr. andSachs, D. H., Fundamental Immunology, 2nd ed., W. E. Paul ed., RavenPress, NY, 889-992 (1989). A suitable procedure is described in detailin Current Protocols in Immunology, above, unit 4.4. Other transplantrejection models which can be used to test the compounds of theinvention are the allogeneic heart transplant models described byTanabe, M. et al., Transplantation, 58:23 (1994) and Tinubu, S. A. etal., J. Immunol., 4330-4338 (1994).

Animal models for delayed type hypersensitivity provides an assay ofcell mediated immune function as well. Delayed type hypersensitivityreactions are a T cell mediated in vivo immune response characterized byinflammation which does not reach a peak until after a period of timehas elapsed after challenge with an antigen. These reactions also occurin tissue specific autoimmune diseases such as multiple sclerosis (MS)and experimental autoimmune encephalomyelitis (EAE, a model for MS). Asuitable procedure is described in detail in Current Protocols inImmunology, above, unit 4.5.

EAE is a T cell mediated autoimmune disease characterized by T cell andmononuclear cell inflammation and subsequent demyelination of axons inthe central nervous system. EAE is generally considered to be a relevantanimal model for MS in humans. Bolton, C., Multiple Sclerosis, 1:143(1995). Both acute and relapsing-remitting models have been developed.The compounds of the invention can be tested for T cell stimulatory orinhibitory activity against immune mediated demyelinating disease usingthe protocol described in Current Protocols in Immunology, above, units15.1 and 15.2. See also the models for myelin disease in whicholigodendrocytes or Schwann cells are grafted into the central nervoussystem as described in Duncan, I. D. et al., Molec. Med. Today, 554-561(1997).

Contact hypersensitivity is a simple delayed type hypersensitivity invivo assay of cell mediated immune function. In this procedure,cutaneous exposure to exogenous haptens which gives rise to a delayedt_(y)pe hypersensitivity reaction which is measured and quantitated.Contact sensitivity involves an initial sensitizing phase followed by anelicitation phase. The elicitation phase occurs when the T lymphocytesencounter an antigen to which they have had previous contact. Swellingand inflammation occur, making this an excellent model of human allergiccontact dermatitis. A suitable procedure is described in detail inCurrent Protocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D.H. Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc.,unit 4.2 (1994). I also Grabbe, S. and Schwarz, T, Immun. Today, 19 (1):37-44 (1998) .

An animal model for arthritis is collagen-induced arthritis. This modelshares clinical, histological and immunological characteristics of humanautoimmune rheumatoid arthritis and is an acceptable model for humanautoimmune arthritis. Mouse and rat models are characterized bysynovitis, erosion of cartilage and subchondral bone. The compounds ofthe invention can be tested for activity against autoimmune arthritisusing the protocols described in Current Protocols in Immunology, above,units 15.5. See also the model using a monoclonal antibody to CD18 andVLA-4 integrins described in Issekutz, A. C. et al , Immunology, 88:569(1996).

A model of asthma has been described in which antigen-induced airwayhyper-reactivity, pulmonary eosinophilia and inflammation are induced bysensitizing an animal with ovalbumin and then challenging the animalwith the same protein delivered by aerosol. Several animal models(guinea pig, rat, non-human primate) show symptoms similar to atopicasthma in humans upon challenge with aerosol antigens. Murine modelshave many of the features of human asthma. Suitable procedures to testthe compounds of the invention for activity and effectiveness in thetreatment of asthma are described by Wolyniec, W. W. et al., Am. J.Respir. Cell Mol. Biol., 18:777 (1998) and the references cited therein.

Additionally, the compounds of the invention can be tested on animalmodels for psoriasis like diseases. Evidence suggests a T cellpathogenesis for psoriasis. The compounds of the invention can be testedin the scid/scid mouse model described by Schon, M. P. et al., Nat.Med., 3:183 (1997), in which the mice demonstrate histopathologic skinlesions resembling psoriasis. Another suitable model is the humanskin/scid mouse chimera prepared as described by Nickoloff, B. J. etal., Am. J. Path., 146:580 (1995).

Recombinant (transgenic) animal models can be engineered by introducingthe coding portion of the genes identified herein into the genome ofanimals of interest, using standard techniques for producing transgenicanimals. Animals that can serve as a target for transgenic manipulationinclude, without limitation, mice, rats, rabbits, guinea pigs, sheep,goats, pigs, and non-human primates, e.g., baboons, chimpanzees andmonkeys. Techniques known in the art to introduce a transgene into suchanimals include pronucleic microinjection (Hoppe and Wanger, U.S. Pat.No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g.,Van der Putten et al., Proc. Natl. Acad. Sci. USA , 82, 6148-615[1985]); gene targeting in embryonic stem cells (Thompson et al., Cell56, 313-321 [1989]); electroporation of embryos (Lo, Mol. Cel. Biol., 3,1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al., Cell57, 717-73 [1989]). For review, see, for example, U.S. Pat. No.4,736,866.

For the purpose of the present invention, transgenic animals includethose that carry the transgene only in part of their cells (“mosaicanimals”). The transgene can be integrated either as a single transgene,or in concatamers, e.g., head-to-head or head-to-tail tandems. Selectiveintroduction of a transgene into a particular cell type is also possibleby following, for example, the technique of Lasko et al., Proc. Natl.Acad. Sci. USA, 89, 6232-636 (1992).

The expression of the transgene in transgenic animals can be monitoredby standard techniques. For example, Southern blot analysis or PCRamplification can be used to verify the integration of the transgene.The level of mRNA expression can then be analyzed using techniques suchas in situ hybridization, Northern blot analysis, PCR, orimmunocytochemistry.

The animals may be further examined for signs of immune diseasepathology, for example by histological examination to determineinfiltration of immune cells into specific tissues. Blocking experimentscan also be performed in which the transgenic animals are treated withthe compounds of the invention to determine the extent of the T cellproliferation stimulation or inhibition of the compounds. In theseexperiments, blocking antibodies which bind to the PRO polypeptide,prepared as described above, are administered to the animal and theeffect on immune function is determined.

Alternatively, “knock out” animals can be constructed which have adefective or altered gene encoding a polypeptide identified herein, as aresult of homologous recombination between the endogenous gene encodingthe polypeptide and altered genomic DNA encoding the same polypeptideintroduced into an embryonic cell of the animal. For example, cDNAencoding a particular polypeptide can be used to clone genomic DNAencoding that polypeptide in accordance with established techniques. Aportion of the genomic DNA encoding a particular polypeptide can bedeleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the polypeptide.

J. ImmunoAdjuvant Therapy

In one embodiment, the immunostimulating compounds of the invention canbe used in immunoadjuvant therapy for the treatment of tumors (cancer).It is now well established that T cells recognize human tumor specificantigens. One group of tumor antigens, encoded by the MAGE, BAGE andGAGE families of genes, are silent in all adult normal tissues , but areexpressed in significant amounts in tumors, such as melanomas, lungtumors, head and neck tumors, and bladder carcinomas. DeSmet, C. et al.,Proc. Natl. Acad. Sci. USA, 93:7149 (1996). It has been shown thatcostimulation of T cells induces tumor regression and an antitumorresponse both in vitro and in vivo. Melero, I. et al., Nature Medicine,3:682 (1997); Kwon, E. D. et al., Proc. Natl. Acad. Sci. USA, 94: 8099(1997); Lynch, D. H. et al., Nature Medicine 3:625 (1997); Finn, 0. J.and Lotze, M. T., J. Immunol., 21:114 (1998). The stimulatory compoundsof the invention can be administered as adjuvants, alone or togetherwith a growth regulating agent, cytotoxic agent or chemotherapeuticagent, to stimulate T cell proliferation/activation and an antitumorresponse to tumor antigens. The growth regulating, cytotoxic, orchemotherapeutic agent may be administered in conventional amounts usingknown administration regimes. Immunostimulating activity by thecompounds of the invention allows reduced amounts of the growthregulating, cytotoxic, or chemotherapeutic agents thereby potentiallylowering the toxicity to the patient.

K. Screening Assays for Drug Candidates

Screening assays for drug candidates are designed to identify compoundsthat bind to or complex with the polypeptides encoded by the genesidentified herein or a biologically active fragment thereof, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds, including peptides, preferably soluble peptides,(poly)peptide-immunoglobulin fusions, and, in particular, antibodiesincluding, without limitation, poly- and monoclonal antibodies andantibody fragments, single-chain antibodies, anti-idiotypic antibodies,and chimeric or humanized versions of such antibodies or fragments, aswell as human antibodies and antibody fragments. The assays can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays and cell basedassays, which are well characterized in the art. All assays are commonin that they call for contacting the drug candidate with a polypeptideencoded by a nucleic acid identified herein under conditions and for atime sufficient to allow these two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the polypeptide encoded by the gene identified herein or thedrug candidate is immobilized on a solid phase, e.g., on a microtiterplate, by covalent or non-covalent attachments. Non-covalent attachmentgenerally is accomplished by coating the solid surface with a solutionof the polypeptide and drying. Alternatively, an immobilized antibody,e.g., a monoclonal antibody, specific for the polypeptide to beimmobilized can be used to anchor it to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g., the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g., by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labelledantibody specifically binding the immobilized complex.

If the candidate compound interacts with but does not bind to aparticular protein encoded by a gene identified herein, its interactionwith that protein can be assayed by methods well known for detectingprotein-protein interactions. Such assays include traditionalapproaches, such as, cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers [Fieldsand Song, Nature (London), 340, 245-246 (1989); Chien et al., Proc.Natl. Acad. Sci. USA, 88, 9578-9582 (1991)] as disclosed by Chevray andNathans, Proc. Natl. Acad. Sci. USA, 89, 5789-5793 (1991). Manytranscriptional activators, such as yeast GAL4, consist of twophysically discrete modular domains, one acting as the DNA-bindingdomain, while the other one functioning as the transcription activationdomain. The yeast expression system described in the foregoingpublications (generally referred to as the “two-hybrid system”) takesadvantage of this property, and employs two hybrid proteins, one inwhich the target protein is fused to the DNA-binding domain of GAL4, andanother, in which candidate activating proteins are fused to theactivation domain. The expression of a GAL1-lacZ reporter gene undercontrol of a GAL4-activated promoter depends on reconstitution of GAL4activity via protein-protein interaction. Colonies containinginteracting polypeptides are detected with a chromogenic substrate for∃-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

In order to find compounds that interfere with the interaction of a geneidentified herein and other intra- or extracellular components can betested, a reaction mixture is usually prepared containing the product ofthe gene and the intra- or extracellular component under conditions andfor a time allowing for the interaction and binding of the two products.To test the ability of a test compound to inhibit binding, the reactionis run in the absence and in the presence of the test compound. Inaddition, a placebo may be added to a third reaction mixture, to serveas positive control. The binding (complex formation) between the testcompound and the intra- or extracellular component present in themixture is monitored as described above. The formation of a complex inthe control reaction(s) but not in the reaction mixture containing thetest compound indicates that the test compound interferes with theinteraction of the test compound and its reaction partner.

L. Compositions and Methods for the Treatment of Immune Related Diseases

The compositions useful in the treatment of immune related diseasesinclude, without limitation, proteins, antibodies, small organicmolecules, peptides, phosphopeptides, antisense and ribozyme molecules,triple helix molecules, etc. that inhibit or stimulate immune function,for example, T cell proliferation/activation, lymphokine release, orimmune cell infiltration.

For example, antisense RNA and RNA molecules act to directly block thetranslation of mRNA by hybridizing to targeted mRNA and preventingprotein translation. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA.

Ribozymes act by sequence-specific hybridization to the complementarytarget RNA, followed by endonucleolytic cleavage. Specific ribozymecleavage sites within a potential RNA target can be identified by knowntechniques. For further details see, e.g., Rossi, Current Biology, 4,469-471 (1994), and PCT publication No. WO 97/33551 (published Sep. 18,1997).

Nucleic acid molecules in triple helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of purines orpyrimidines on one strand of a duplex. For further details see, e.g.,PCT publication No. WO 97/33551, supra.

These molecules can be identified by any or any combination of thescreening assays discussed above and/or by any other screeningtechniques well known for those skilled in the art.

M. Anti-PRO Antibodies

The present invention further provides anti-PRO antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-PRO antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the PRO polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-PRO antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the PRO polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103] Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against PRO.Preferably, the binding specificity of monoclonal antibodies produced bythe hybridoma cells is determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Human and Humanized Antibodies

The anti-PRO antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al., and Boerner et al., are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368: 812-13 (1994); Fishwild et al., Nature Biotechnology, 14: 845-51(1996); Neuberger, Nature Biotechnology, 14: 826 (1996); Lonberg andHuszar, Intern. Rev. Immunol., 13: 65-93 (1995).

The antibodies may also be affinity matured using known selection and/ormutagenesis methods as described above. Preferred affinity maturedantibodies have an affinity which is five times, more preferably 10times, even more preferably 20 or 30 times greater than the startingantibody (generally murine, humanized or human) from which the maturedantibody is prepared.

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe PRO, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g., F(ab′)2 bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al.,Science, 229:81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab' fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.,175:217-225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)2 molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various technique for making and isolating bispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bispecific antibodies have been produced using leucine zippers.Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab'portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. This method can also beutilized for the production of antibody homodimers. The “diabody”technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA,90:6444-6448 (1993) has provided an alternative mechanism for makingbispecific antibody fragments. The fragments comprise a heavy-chainvariable domain (VH) connected to a light-chain variable domain (VL) bya linker which is too short to allow pairing between the two domains onthe same chain. Accordingly, the VH and VL domains of one fragment areforced to pair with the complementary VL and VH domains of anotherfragment, thereby forming two antigen-binding sites. Another strategyfor making bispecific antibody fragments by the use of single-chain Fv(sFv) dimers has also been reported. See, Gruber et al., J. Immunol.152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven PRO polypeptide herein. Alternatively, an anti-PRO polypeptide armmay be combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g., CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcgR), such as FcgRI (CD64), FcgRII (CD32)and FcgRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular PRO polypeptide. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which express aparticular PRO polypeptide. These antibodies possess a PRO-binding armand an arm which binds a cytotoxic agent or a radionuclide chelator,such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody ofinterest binds the PRO polypeptide and further binds tissue factor (TF).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al., CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 23:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19):1484 (1989).

9. Uses for Anti-PRO Antibodies

The anti-PRO antibodies of the invention have various utilities. Forexample, anti-PRO antibodies may be used in diagnostic assays for PRO,e.g., detecting its expression (and in some cases, differentialexpression) in specific cells, tissues, or serum. Various diagnosticassay techniques known in the art may be used, such as competitivebinding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-PRO antibodies also are useful for the affinity purification of PROfrom recombinant cell culture or natural sources. In this process, theantibodies against PRO are immobilized on a suitable support, such aSephadex resin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing the PROto be purified, and thereafter the support is washed with a suitablesolvent that will remove substantially all the material in the sampleexcept the PRO, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent that will release thePRO from the antibody.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

N. Pharmaceutical Compositions

The active PRO molecules of the invention (e.g., PRO polypeptides,anti-PRO antibodies, and/or variants of each) as well as other moleculesidentified by the screening assays disclosed above, can be administeredfor the treatment of immune related diseases, in the form ofpharmaceutical compositions. Therapeutic formulations of the active PROmolecule, preferably a polypeptide or antibody of the invention, areprepared for storage by mixing the active molecule having the desireddegree of purity with optional pharmaceutically acceptable carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. [1980]), in the form of lyophilized formulationsor aqueous solutions. Acceptable carriers, excipients, or stabilizersare nontoxic to recipients at the dosages and concentrations employed,and include buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Compounds identified by the screening assays disclosed herein can beformulated in an analogous manner, using standard techniques well knownin the art.

Lipofections or liposomes can also be used to deliver the PRO moleculeinto cells. Where antibody fragments are used, the smallest inhibitoryfragment which specifically binds to the binding domain of the targetprotein is preferred. For example, based upon the variable regionsequences of an antibody, peptide molecules can be designed which retainthe ability to bind the target protein sequence. Such peptides can besynthesized chemically and/or produced by recombinant DNA technology(see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA. 90:7889-7893[1993]).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise a cytotoxicagent, cytokine or growth inhibitory agent. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended.

The active PRO molecules may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations or the PRO molecules may be prepared.Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and (-ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S-S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

O. Methods of Treatment

It is contemplated that the polypeptides, antibodies and other activecompounds of the present invention may be used to treat various immunerelated diseases and conditions, such as T cell mediated diseases,including those characterized by infiltration of inflammatory cells intoa tissue, stimulation of T-cell proliferation, inhibition of T-cellproliferation, increased or decreased vascular permeability or theinhibition thereof.

Exemplary conditions or disorders to be treated with the polypeptides,antibodies and other compounds of the invention, include, but are notlimited to systemic lupus erythematosis, rheumatoid arthritis, juvenilechronic arthritis, osteoarthritis, spondyloarthropathies, systemicsclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease (ulcerative colitis: Crohn's disease),gluten-sensitive enteropathy, and Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonia, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft -versus-host-disease.

In systemic lupus erythematosus, the central mediator of disease is theproduction of auto-reactive antibodies to self proteins/tissues and thesubsequent generation of immune-mediated inflammation. Antibodies eitherdirectly or indirectly mediate tissue injury. Though T lymphocytes havenot been shown to be directly involved in tissue damage, T lymphocytesare required for the development of auto-reactive antibodies. Thegenesis of the disease is thus T lymphocyte dependent. Multiple organsand systems are affected clinically including kidney, lung,musculoskeletal system, mucocutaneous, eye, central nervous system,cardiovascular system, gastrointestinal tract, bone marrow and blood.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatorydisease that mainly involves the synovial membrane of multiple jointswith resultant injury to the articular cartilage. The pathogenesis is Tlymphocyte dependent and is associated with the production of rheumatoidfactors, auto-antibodies directed against self IgG, with the resultantformation of immune complexes that attain high levels in joint fluid andblood. These complexes in the joint may induce the marked infiltrate oflymphocytes and monocytes into the synovium and subsequent markedsynovial changes; the joint space/fluid if infiltrated by similar cellswith the addition of numerous neutrophils. Tissues affected areprimarily the joints, often in symmetrical pattern. However,extra-articular disease also occurs in two major forms. One form is thedevelopment of extra-articular lesions with ongoing progressive jointdisease and typical lesions of pulmonary fibrosis, vasculitis, andcutaneous ulcers. The second form of extra-articular disease is the socalled Felty's syndrome which occurs late in the RA disease course,sometimes after joint disease has become quiescent, and involves thepresence of neutropenia, thrombocytopenia and splenomegaly. This can beaccompanied by vasculitis in multiple organs with formations ofinfarcts, skin ulcers and gangrene. Patients often also developrheumatoid nodules in the subcutis tissue overlying affected joints; thenodules late stage have necrotic centers surrounded by a mixedinflammatory cell infiltrate. Other manifestations which can occur in RAinclude: pericarditis, pleuritis, coronary arteritis, interstitialpneumonitis with pulmonary fibrosis, keratoconjunctivitis sicca, andrheumatoid nodules.

Juvenile chronic arthritis is a chronic idiopathic inflammatory diseasewhich begins often at less than 16 years of age. Its phenotype has somesimilarities to RA; some patients which are rheumatoid factor positiveare classified as juvenile rheumatoid arthritis. The disease issub-classified into three major categories: pauciarticular,polyarticular, and systemic. The arthritis can be severe and istypically destructive and leads to joint ankylosis and retarded growth.Other manifestations can include chronic anterior uveitis and systemicamyloidosis.

Spondyloarthropathies are a group of disorders with some common clinicalfeatures and the common association with the expression of HLA-B27 geneproduct. The disorders include: ankylosing spondylitis, Reiter'ssyndrome (reactive arthritis), arthritis associated with inflammatorybowel disease, spondylitis associated with psoriasis, juvenile onsetspondyloarthropathy and undifferentiated spondyloarthropathy.Distinguishing features include sacroileitis with or withoutspondylitis; inflammatory asymmetric arthritis; association with HLA-B27(a serologically defined allele of the HLA-B locus of class I MHC);ocular inflammation, and absence of autoantibodies associated with otherrheumatoid disease. The cell most implicated as key to induction of thedisease is the CD8⁺ T lymphocyte, a cell which targets antigen presentedby class I MHC molecules. CD8⁺ T cells may react against the class I MHCallele HLA-B27 as if it were a foreign peptide expressed by MHC class Imolecules. It has been hypothesized that an epitope of HLA-B27 may mimica bacterial or other microbial antigenic epitope and thus induce a CD8⁺T cells response.

Systemic sclerosis (scleroderma) has an unknown etiology. A hallmark ofthe disease is induration of the skin; likely this is induced by anactive inflammatory process. Scleroderma can be localized or systemic;vascular lesions are common and endothelial cell injury in themicrovasculature is an early and important event in the development ofsystemic sclerosis; the vascular injury may be immune mediated. Animmunologic basis is implied by the presence of mononuclear cellinfiltrates in the cutaneous lesions and the presence of anti-nuclearantibodies in many patients. ICAM-1 is often upregulated on the cellsurface of fibroblasts in skin lesions suggesting that T cellinteraction with these cells may have a role in the pathogenesis of thedisease. Other organs involved include: the gastrointestinal tract:smooth muscle atrophy and fibrosis resulting in abnormalperistalsis/motility; kidney: concentric subendothelial intimalproliferation affecting small arcuate and interlobular arteries withresultant reduced renal cortical blood flow, results in proteinuria,azotemia and hypertension; skeletal muscle: atrophy, interstitialfibrosis; inflammation; lung: interstitial pneumonitis and interstitialfibrosis; and heart: contraction band necrosis, scarring/fibrosis.

Idiopathic inflammatory myopathies including dermatomyositis,polymyositis and others are disorders of chronic muscle inflammation ofunknown etiology resulting in muscle weakness. Muscleinjury/inflammation is often symmetric and progressive. Autoantibodiesare associated with most forms. These myositis-specific autoantibodiesare directed against and inhibit the function of components, proteinsand RNA's, involved in protein synthesis.

Sjögren's syndrome is due to immune-mediated inflammation and subsequentfunctional destruction of the tear glands and salivary glands. Thedisease can be associated with or accompanied by inflammatory connectivetissue diseases. The disease is associated with autoantibody productionagainst Ro and La antigens, both of which are small RNA-proteincomplexes. Lesions result in keratoconjunctivitis sicca, xerostomia,with other manifestations or associations including biliary cirrhosis,peripheral or sensory neuropathy, and palpable purpura.

Systemic vasculitis are diseases in which the primary lesion isinflammation and subsequent damage to blood vessels which results inischemia/necrosis/degeneration to tissues supplied by the affectedvessels and eventual end-organ dysfunction in some cases. Vasculitidescan also occur as a secondary lesion or sequelae to otherimmune-inflammatory mediated diseases such as rheumatoid arthritis,systemic sclerosis, etc., particularly in diseases also associated withthe formation of immune complexes. Diseases in the primary systemicvasculitis group include: systemic necrotizing vasculitis: polyarteritisnodosa, allergic angiitis and granulomatosis, polyangiitis; Wegener'sgranulomatosis; lymphomatoid granulomatosis; and giant cell arteritis.Miscellaneous vasculitides include: mucocutaneous lymph node syndrome(MLNS or Kawasaki's disease), isolated CNS vasculitis, Behet's disease,thromboangiitis obliterans (Buerger's disease) and cutaneous necrotizingvenulitis. The pathogenic mechanism of most of the types of vasculitislisted is believed to be primarily due to the deposition ofimmunoglobulin complexes in the vessel wall and subsequent induction ofan inflammatory response either via ADCC, complement activation, orboth.

Sarcoidosis is a condition of unknown etiology which is characterized bythe presence of epithelioid granulomas in nearly any tissue in the body;involvement of the lung is most common. The pathogenesis involves thepersistence of activated macrophages and lymphoid cells at sites of thedisease with subsequent chronic sequelae resultant from the release oflocally and systemically active products released by these cell types.

Autoimmune hemolytic anemia including autoimmune hemolytic anemia,immune pancytopenia, and paroxysmal noctural hemoglobinuria is a resultof production of antibodies that react with antigens expressed on thesurface of red blood cells (and in some cases other blood cellsincluding platelets as well) and is a reflection of the removal of thoseantibody coated cells via complement mediated lysis and/orADCC/Fc-receptor-mediated mechanisms.

In autoimmune thrombocytopenia including thrombocytopenic purpura, andimmune-mediated thrombocytopenia in other clinical settings, plateletdestruction/removal occurs as a result of either antibody or complementattaching to platelets and subsequent removal by complement lysis, ADCCor FC-receptor mediated mechanisms.

Thyroiditis including Grave's disease, Hashimoto's thyroiditis, juvenilelymphocytic thyroiditis, and atrophic thyroiditis, are the result of anautoimmune response against thyroid antigens with production ofantibodies that react with proteins present in and often specific forthe thyroid gland. Experimental models exist including spontaneousmodels: rats (BUF and BB rats) and chickens (obese chicken strain);inducible models: immunization of animals with either thyroglobulin,thyroid microsomal antigen (thyroid peroxidase).

Type I diabetes mellitus or insulin-dependent diabetes is the autoimmunedestruction of pancreatic islet cells; this destruction is mediated byauto-antibodies and auto-reactive T cells. Antibodies to insulin or theinsulin receptor can also produce the phenotype ofinsulin-non-responsiveness.

Immune mediated renal diseases, including glomerulonephritis andtubulointerstitial nephritis, are the result of antibody or T lymphocytemediated injury to renal tissue either directly as a result of theproduction of autoreactive antibodies or T cells against renal antigensor indirectly as a result of the deposition of antibodies and/or immunecomplexes in the kidney that are reactive against other, non-renalantigens. Thus other immune-mediated diseases that result in theformation of immune-complexes can also induce immune mediated renaldisease as an indirect sequelae. Both direct and indirect immunemechanisms result in inflammatory response that produces/induces lesiondevelopment in renal tissues with resultant organ function impairmentand in some cases progression to renal failure. Both humoral andcellular immune mechanisms can be involved in the pathogenesis oflesions.

Demyelinating diseases of the central and peripheral nervous systems,including multiple sclerosis;

idiopathic demyelinating polyneuropathy or Guillain-Barré syndrome; andchronic inflammatory demyelinating polyneuropathy, are believed to havean autoimmune basis and result in nerve demyelination as a result ofdamage caused to oligodendrocytes or to myelin directly. In MS there isevidence to suggest that disease induction and progression is dependenton T lymphocytes. Multiple sclerosis is a demyelinating disease that isT lymphocyte-dependent and has either a relapsing-remitting course or achronic progressive course. The etiology is unknown; however, viralinfections, genetic predisposition, environment, and autoimmunity allcontribute. Lesions contain infiltrates of predominantly T lymphocytemediated, microglial cells and infiltrating macrophages; CD4⁺Tlymphocytes are the predominant cell type at lesions. The mechanism ofoligodendrocyte cell death and subsequent demyelination is not known butis likely T lymphocyte driven.

Inflammatory and fibrotic lung disease, including eosinophilicpneumonia; idiopathic pulmonary fibrosis, and hypersensitivitypneumonitis may involve a disregulated immune-inflammatory response.Inhibition of that response would be of therapeutic benefit.

Autoimmune or immune-mediated skin disease including bullous skindiseases, erythema multiforme, and contact dermatitis are mediated byauto-antibodies, the genesis of which is T lymphocyte-dependent.

Psoriasis is a T lymphocyte-mediated inflammatory disease. Lesionscontain infiltrates of T lymphocytes, macrophages and antigen processingcells, and some neutrophils.

Allergic diseases, including asthma; allergic rhinitis; atopicdermatitis; food hypersensitivity; and urticaria are T lymphocytedependent. These diseases are predominantly mediated by T lymphocyteinduced inflammation, IgE mediated-inflammation or a combination ofboth.

Transplantation associated diseases, including graft rejection andgraft-versus-host-disease (GVHD) are T lymphocyte-dependent; inhibitionof T lymphocyte function is ameliorative.

Other diseases in which intervention of the immune and/or inflammatoryresponse have benefit are infectious disease including but not limitedto viral infection (including but not limited to AIDS, hepatitis A, B,C, D, E and herpes) bacterial infection, fungal infections, andprotozoal and parasitic infections (molecules (or derivatives/agonists)which stimulate the MLR can be utilized therapeutically to enhance theimmune response to infectious agents), diseases of immunodeficiency(molecules/derivatives/agonists) which stimulate the MLR can be utilizedtherapeutically to enhance the immune response for conditions ofinherited, acquired, infectious induced (as in HIV infection), oriatrogenic (i.e., as from chemotherapy) immunodeficiency, and neoplasia.

It has been demonstrated that some human cancer patients develop anantibody and/or T lymphocyte response to antigens on neoplastic cells.It has also been shown in animal models of neoplasia that enhancement ofthe immune response can result in rejection or regression of thatparticular neoplasm. Molecules that enhance the T lymphocyte response inthe MLR have utility in vivo in enhancing the immune response againstneoplasia. Molecules which enhance the T lymphocyte proliferativeresponse in the MLR (or small molecule agonists or antibodies thataffected the same receptor in an agonistic fashion) can be usedtherapeutically to treat cancer. Molecules that inhibit the lymphocyteresponse in the MLR also function in vivo during neoplasia to suppressthe immune response to a neoplasm; such molecules can either beexpressed by the neoplastic cells themselves or their expression can beinduced by the neoplasm in other cells. Antagonism of such inhibitorymolecules (either with antibody, small molecule antagonists or othermeans) enhances immune-mediated tumor rejection.

Additionally, inhibition of molecules with proinflammatory propertiesmay have therapeutic benefit in reperfusion injury; stroke; myocardialinfarction; atherosclerosis; acute lung injury; hemorrhagic shock; burn;sepsis/septic shock; acute tubular necrosis; endometriosis; degenerativejoint disease and pancreatitis. The compounds of the present invention,e.g., polypeptides or antibodies, are administered to a mammal,preferably a human, in accord with known methods, such as intravenousadministration as a bolus or by continuous infusion over a period oftime, by intramuscular, intraperitoneal, intracerebral spinal,subcutaneous, intra-articular, intra synovial, intrathecal, oral,topical, or inhalation (intranasal, intrapulmonary) routes. Intravenousor inhaled administration of polypeptides and antibodies is preferred.In immunoadjuvant therapy, other therapeutic regimens, suchadministration of an anti-cancer agent, may be combined with theadministration of the proteins, antibodies or compounds of the instantinvention. For example, the patient to be treated with a theimmunoadjuvant of the invention may also receive an anti-cancer agent(chemotherapeutic agent) or radiation therapy. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service, Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede,or follow administration of the immunoadjuvant or may be givensimultaneously therewith. Additionally, an anti-oestrogen compound suchas tamoxifen or an anti-progesterone such as onapristone (see, EP616812) may be given in dosages known for such molecules. It may bedesirable to also administer antibodies against other immune diseaseassociated or tumor associated antigens, such as antibodies which bindto CD20, CD11a, CD18, ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelialfactor (VEGF). Alternatively, or in addition, two or more antibodiesbinding the same or two or more different antigens disclosed herein maybe coadministered to the patient. Sometimes, it may be beneficial toalso administer one or more cytokines to the patient. In one embodiment,the PRO polypeptides are coadministered with a growth inhibitory agent.For example, the growth inhibitory agent may be administered first,followed by a PRO polypeptide. However, simultaneous administration oradministration first is also contemplated. Suitable dosages for thegrowth inhibitory agent are those presently used and may be lowered dueto the combined action (synergy) of the growth inhibitory agent and thePRO polypeptide. For the treatment or reduction in the severity ofimmune related disease, the appropriate dosage of an a compound of theinvention will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the agent isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the compound, and thediscretion of the attending physician. The compound is suitablyadministered to the patient at one time or over a series of treatments.

For example, depending on the type and severity of the disease, about 1mg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of polypeptide or antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 mg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

P. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials (e.g., comprising a PRO molecule) useful for thediagnosis or treatment of the disorders described above is provided. Thearticle of manufacture comprises a container and an instruction.Suitable containers include, for example, bottles, vials, syringes, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition which iseffective for diagnosing or treating the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agent in the composition is usually apolypeptide or an antibody of the invention. An instruction or label on,or associated with, the container indicates that the composition is usedfor diagnosing or treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

Q. Diagnosis and Prognosis of Immune Related Disease

Cell surface proteins, such as proteins which are overexpressed incertain immune related diseases, are excellent targets for drugcandidates or disease treatment. The same proteins along with secretedproteins encoded by the genes amplified in immune related disease statesfind additional use in the diagnosis and prognosis of these diseases.For example, antibodies directed against the protein products of genesamplified in multiple sclerosis, rheumatoid arthritis, inflammatorybowel disorder, or another immune related disease, can be used asdiagnostics or prognostics.

For example, antibodies, including antibody fragments, can be used toqualitatively or quantitatively detect the expression of proteinsencoded by amplified or overexpressed genes (“marker gene products”).The antibody preferably is equipped with a detectable, e.g., fluorescentlabel, and binding can be monitored by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. These techniques areparticularly suitable, if the overexpressed gene encodes a cell surfaceprotein Such binding assays are performed essentially as describedabove.

In situ detection of antibody binding to the marker gene products can beperformed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent for those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Isolation of cDNA Clones Encoding Human PRO1031

The extracellular domain (ECD) sequences (including the secretionsignal, if any) of from about 950 known secreted proteins from theSwiss-Prot public protein database were used to search expressedsequence tag (EST) databases. The EST databases included public ESTdatabases (e.g., GenBank, Merck/Wash U.) and a proprietary EST DNAdatabase (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.). Thesearch was performed using the computer program BLAST or BLAST2 (Altshulet al., Methods in Enzymology, 266:460-480 (1996)) as a comparison ofthe ECD protein sequences to a 6 frame translation of the EST sequence.Those comparisons resulting in a BLAST score of 70 (or in some cases,90) or greater that did not encode known proteins were clustered andassembled into consensus DNA sequences with the program “phrap” (PhilGreen, University of Washington, Seattle, Wash.).

An initial virtual sequence fragment (consensus assembly) was assembledrelative to other EST sequences using phrap. The initial consensus DNAsequence was extended using repeated cycles of BLAST and phrap to extendthe consensus sequence as far as possible using the sources of ESTsequences discussed above. The results of this consensus assembly isreferred to as DNA47332.

One sequence comprising the consensus assembly, W74558 (clone 344649)was further examined. The sequence was obtained from the IMAGEconsortium and analyzed. Lennon et al., Genomics 33: 151 (1996). DNAsequencing gave the full-length DNA sequence for PRO1031 [hereindesignated as DNA59294-1381] (SEQ ID NO:1) and the derived PRO1031protein sequence (UNQ516)(SEQ ID NO: 2).

The entire nucleotide sequence of DNA59294-1381 is shown in FIG. 1 (SEQID NO:1). Clone DNA59294-1381 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 42-44and ending at the stop codon at nucleotide positions 582-584 (FIG. 1;SEQ ID NO:1). The predicted polypeptide precursor is 180 amino acidslong (FIG. 2; SEQ ID NO:2). The full-length PRO1031 (UNQ516) proteinshown in FIG. 2 (SEQ ID NO:2) has an estimated molecular weight of about20,437 daltons and a pI of about 9.58. Clone DNA59294-1381 has beendeposited with the ATCC, and has been assigned deposit number 209866. Inthe event of any sequencing irregularities or errors with the sequencesprovided herein, it is understood that the deposited clone contains thecorrect sequence for DNA59624-1381 (SEQ ID NO:1). Furthermore, thesequences provided herein are the result of known sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO1031polypeptide (UNQ516)(SEQ ID NO:2) suggests that it is a novelinterleukin-17 homolog, herein designated as IL-17B.

Further analysis of the amino acid sequence of SEQ ID NO:2 reveals thatthe putative signal peptide is at about amino acids 1-20 of SEQ ID NO:2.An N-glycosylation site is at about amino acids 75-78 of SEQ ID NO:2. Aregion having sequence identity with IL-17 is at about amino acids96-180. The corresponding nucleotides can be routinely determined giventhe sequences provided herein.

Example 2 Isolation of cDNA clones Encoding Human PRO1122

An expressed sequence tag (EST) DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.) was searched and an EST wasidentified. The EST was Incyte 1347523 also called DNA49665. Based onDNA49665, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolated a clone of the full-length coding sequence for thePRO1122. [e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual(New York: Cold Spring Harbor Laboratory Press, 1989); Dieffenbach etal., PCR Primer: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, 1995)].

Forward and reverse PCR primers generally range from 20 to 30nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probes sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kpb. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

PCR primers (forward, reverse and hybridization) were synthesized:

forward PCR primer: (SEQ ID NO: 19) 5′-ATCCACAGAAGCTGGCCTTCGCCG-3′reverse PCR primer: (SEQ ID NO: 20) 5′-GGGACGTGGATGAACTCGGTGTGG-3′hybridization probe: (SEQ ID NO: 21)5′-TATCCACAGAAGCTGGCCTTCGCCGAGTGCCTGTGCAGAG-3′.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO1122 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed using standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science 235:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1122 [herein designated asDNA62377-1381-1](SEQ ID NO:3) and the derived protein PRO1122 sequence(UNQ561)(SEQ ID NO:4).

The entire nucleotide sequence of DNA62377-1381-1 (SEQ ID NO:3) is shownin FIG. 3 (SEQ ID NO:3). Clone DNA62377-1381-1 (SEQ ID NO:3) contains asingle open reading frame with an apparent translational initiation siteat nucleotide positions 50-52 and ending at the stop codon at nucleotidepositions 641-643 of SEQ ID NO:3 (FIG. 3). The predicted polypeptideprecursor is 197 amino acids long (FIG. 4; SEQ ID NO:4). The full-lengthPRO1122 protein shown in FIG. 4 (UNQ561)(SEQ ID NO:4) has an estimatedmolecular weight of about 21765 daltons and a pI of about 8.53. CloneDNA62377-1381-1 has been deposited with the ATCC on Dec. 22, 1998 andhas been assigned deposit number 203552. It is understood that in theevent or a sequencing irregularity or error in the sequences providedherein, the correct sequence is the sequence deposited. Furthermore, allsequences provided herein are the result of known sequencing techniques.

Analysis of the amino acid sequence of the isolated full-length PRO1122(UNQ561) suggests that it possesses similarity with IL-17, therebyindicating that PRO1122 (UNQ561) may be a novel cytokine and is hereindesignated IL-17C. FIG. 4 (SEQ ID NO:4) also shows the approximatelocations of the signal peptide, leucine zipper pattern, and a regionhaving sequence identity with IL-17.

Example 3 Isolation of cDNA Clones Encoding Human PRO10272

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search genomic DNA sequencesfrom GenBank. The search was performed using the computer program BLASTor BLAST2 [Altschul et al., Methods in Enzymology, 266:460-480 (1996)]as a comparison of the ECD protein sequences to a 6 frame translation ofthe EST sequences. Those comparisons resulting in a BLAST score of 70(or in some cases, 90) or greater that did not encode known proteinswere clustered and assembled into consensus DNA sequences with theprogram “phrap” (Phil Green, University of Washington, Seattle, Wash.).

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described above. This consensus sequence is hereindesignated DNA146646. In some cases, the consensus sequence derives froman intermediate consensus DNA sequence which was extended using repeatedcycles of BLAST and phrap to extend that intermediate consensus sequenceas far as possible using the sources of EST sequences discussed above.

Based on the DNA146646 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO10272. Forward and reverse PCRprimers generally range from 20 to 30 nucleotides and are often designedto give a PCR product of about 100-1000 bp in length. The probesequences are typically 40-55 bp in length. In some cases, additionaloligonucleotides are synthesized when the consensus sequence is greaterthan about 1-1.5 kbp. In order to screen several libraries for afull-length clone, DNA from the libraries was screened by PCRamplification, as per Ausubel et al., Current Protocols in MolecularBiology, supra, with the PCR primer pair. A positive library was thenused to isolate clones encoding the gene of interest using the probeoligonucleotide and one of the primer pairs. PCR primers (forward andreverse) were synthesized:

forward PCR primer: (SEQ ID NO: 22) 5′-GTTGCATTCTTGGCAATGGTCATGGGA-3′reverse PCR primer: (SEQ ID NO: 23) 5′-GGTCCATGTGGGAGCCTGTCTGTA-3′Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA146646 sequence which had thefollowing nucleotide sequence:

hybridization probe (SEQ ID NO: 24)5′-CAGCAGCTCCTCAGAGGTGTCCTGCCCTTTGCTGGGGCAGCAGCT-3′

RNA for construction of the cDNA libraries was isolated from humantestis tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a full-length PRO10272 polypeptide(designated herein as DNA147531-2821 [FIG. 5, SEQ ID NO: 5]) and thederived protein sequence for that PRO10272 polypeptide.

The full length clone identified above contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 259-261 and a stop signal at nucleotide positions 790-792(FIG. 5, SEQ ID NO:5). The predicted polypeptide precursor is 177 aminoacids long, has a calculated molecular weight of approximately 20,330daltons and an estimated pI of approximately 8.78. Analysis of thefull-length PRO10272 sequence shown in FIG. 6 (SEQ ID NO:6) evidencesthe presence of a variety of important polypeptide domains as shown inFIG. 6, wherein the locations given for those important polypeptidedomains are approximate as described above. Clone DNA147531-2821 hasbeen deposited with ATCC on Jan. 11, 2000 and is assigned ATCC depositno. PTA-1185.

Analysis of the amino acid sequence of the isolated full-length PRO10272suggests that it possesses similarity with IL-17 and various homologs ofit, thereby indicating that PRO10272 may be a novel cytokine and isherein designated IL-17E. Specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35), using the ALIGN-2 sequencealignment analysis of the full-length sequence shown in FIG. 6 (SEQ IDNO:6), evidenced sequence identity between the PRO10272 amino acidsequence and the following Dayhoff sequences: P_Y22197, P_W85620,AF18469_(—)1, P_Y41762, P_Y28235, P_W97350, P_Y22198, P_Y28236,P_W28514, P_W13651.

Example 4 Isolation of cDNA Clones Encoding a Human PRO21175

An expressed sequence tag (EST) DNA database from Merck/WashingtonUniversity was searched and an EST was identified which showed homologyto Interleukin-17.

A pool of 50 different human cDNA libraries from various tissues wasused in cloning. The cDNA libraries used to isolate the cDNA clonesencoding human PRO21175 were constructed by standard methods usingcommercially available reagents such as those from Invitrogen, SanDiego, Calif. The cDNA was primed with oligo dT containing a NotI site,linked with blunt to SalI hemikinased adaptors, cleaved with NotI, sizedappropriately by gel electrophoresis, and cloned in a definedorientation into a suitable cloning vector (such as pRKB or pRKD; pRK5Bis a precursor of pRK5D that does not contain the SfiI site; see, Holmeset al., Science, 253:1278-1280 (1991)) in the unique XhoI and NotI.

Oligonucleotides probes based upon the above described EST sequence werethen synthesized: 1) to identify by PCR a cDNA library that containedthe sequence of interest, and 2) for use as probes to isolate a clone ofthe full-length coding sequence for PRO21175. Forward and reverse PCRprimers generally range from 20 to 30 nucleotides and are often designedto give a PCR product of about 100-1000 bp in length. The probesequences are typically 40-55 bp in length. In order to screen severallibraries for a full-length clone, DNA from the libraries was screenedby PCR amplification, as per Ausubel et al., Current Protocols inMolecular Biology, supra, with the PCR primer pair. A positive librarywas then used to isolate clones encoding the gene of interest using theprobe oligonucleotide and one of the primer pairs.

The oligonucleotide probes employed were as follows:

forward PCR primer (SEQ ID NO: 25) 5′-GCTCAGTGCCTTCCACCACACGC-3′reverse PCR primer (SEQ ID NO: 26) 5′-CTGCGTCCTTCTCCGGCTCGG-3′hybridization probe (SEQ ID NO: 27) 5′CGTTCCGTCTACACCGAGGCCTACGTCACCATCCCCGTGGGCTGC-3′

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 1-3 and a stop signal at nucleotide positions 607-609 (FIG. 7,SEQ ID NO:7). The predicted polypeptide precursor is 202 amino acidslong, has a calculated molecular weight of approximately 21,879 daltonsand an estimated pI of approximately 9.3. Analysis of the full-lengthPRO21175 sequence shown in FIG. 8 (SEQ ID NO:8) evidences the presenceof a variety of important polypeptide domains as shown in FIG. 8,wherein the locations given for those important polypeptide domains areapproximate as described above. Chromosome mapping evidences that thePRO21175-encoding nucleic acid maps to 13q11 in humans. CloneDNA173894-2947 has been deposited with ATCC on Jun. 20, 2000 and isassigned ATCC deposit no. PTA-2108.

Analysis of the amino acid sequence of the isolated full-length PRO21175suggests that it possesses similarity with IL-17, thereby indicatingthat PRO21175 may be a novel cytokine and is herein designated

IL-17D. Specifically, an analysis of the protein database (version 35.45SwissProt 35), using the ALIGN-2 sequence alignment analysis of thefull-length sequence shown in FIG. 8 (SEQ ID NO:8), evidenced sequenceidentity between the PRO21175 amino acid sequence and the followingsequence: AF152099_(—)1.

Example 5 Isolation of cDNA Clones Encoding a Human PRO5801

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included (1) public EST databases (e.g., GenBank) and (2) aproprietary EST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST2 [Altschul et al., Methods in Enzymology, 266:460-480 (1996)] as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons resulting in a BLAST score of 70 (or insome cases, 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described above. This consensus sequence is hereindesignated DNA105850. In some cases, the consensus sequence derives froman intermediate consensus DNA sequence which was extended using repeatedcycles of BLAST and phrap to extend that intermediate consensus sequenceas far as possible using the sources of EST sequences discussed above.

Based on the DNA105850 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO5801. Forward and reverse PCR primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, supra, with the PCR primerpair. A positive library was then used to isolate clones encoding thegene of interest using the probe oligonucleotide and one of the primerpairs.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 1  (SEQ ID NO: 28) 5′-ACTCCATATTTTCCTACTTGTGGCA-3′forward PCR primer 2  (SEQ ID NO: 29) 5′-CCCAAAGTGACCTAAGAAC-3′reverse PCR  (SEQ ID NO: 30) primer 5′-TCACTGAATTTCTTCAAAACCATTGCA-3′Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA105850 sequence which had thefollowing nucleotide sequence

hybridization probe (SEQ ID NO: 31)5′-TGTGGCAGCGACTGCATCCGACATAAAGGAACAGTTGTGCTCTGC CCACA-3′

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a full-length PRO5801 polypeptide(designated herein as DNA115291-2681 [FIG. 11, SEQ ID NO: 11]) and thederived protein sequence for that PRO5801 polypeptide.

The full length clone identified above contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 7-9 and a stop signal at nucleotide positions 1513-1515 (FIG.12, SEQ ID NO:12). The predicted polypeptide precursor is 502 aminoacids long, has a calculated molecular weight of approximately 55,884daltons and an estimated pI of approximately 8.52. Analysis of thefull-length PRO5801 sequence shown in FIG. 12 (SEQ ID NO:12) evidencesthe presence of a variety of important polypeptide domains as shown inFIG. 12, wherein the locations given for those important polypeptidedomains are approximate as described above. Clone DNA115291-2681 hasbeen deposited with ATCC on Jun. 8, 1999 and is assigned ATCC depositno. PTA-202.

An analysis of the Dayhoff database shows that PRO5801 has sequencesimilarity to an IL-17 receptor protein and PRO5801 is also designatedherein as IL-17RH1 as described in Example 22 of the presentapplication. Specifically, an analysis of the Dayhoff database (version35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis ofthe full-length sequence shown in FIG. 12 (SEQ ID NO:12), evidencedsequence identity between the PRO5801 amino acid sequence and thefollowing Dayhoff sequences: HSU58917_(—)1, P_W92409, P_W61272,P_W04185, P_W61271, P_W04184, P_W92408, GEN13979, MMU31993_(—)1 andYSO2_CAEEL.

Example 6 Isolation of cDNA Clones Encoding a Human PRO20040

An expressed sequence tag (EST) DNA database (Merck/WashingtonUniversity) was searched and an EST was identified which showed homologyto Interleukin 17 receptor.

RNA for construction of cDNA libraries was then isolated from a pool of50 different human cDNA libraries. The cDNA libraries used to isolatethe cDNA clones encoding human PRO20040 were constructed by standardmethods using commercially available reagents such as those fromInvitrogen, San Diego, Calif. The cDNA was primed with oligo dTcontaining a NotI site, linked with blunt to SalI hemikinased adaptors,cleaved with NotI, sized appropriately by gel electrophoresis, andcloned in a defined orientation into a suitable cloning vector (such aspRKB or pRKD; pRKSB is a precursor of pRKSD that does not contain theSfiI site; see, Holmes et al., Science, 253:1278-1280 (1991)) in theunique XhoI and NotI.

Oligonucleotides probes based upon the above described EST sequence werethen synthesized: 1) to identify by PCR a cDNA library that containedthe sequence of interest, and 2) for use as probes to isolate a clone ofthe full-length coding sequence for PRO20040. Forward and reverse PCRprimers generally range from 20 to 30 nucleotides and are often designedto give a PCR product of about 100-1000 bp in length. The probesequences are typically 40-55 bp in length. In order to screen severallibraries for a full-length clone, DNA from the libraries was screenedby PCR amplification, as per Ausubel et al., Current Protocols inMolecular Biology, supra, with the PCR primer pair. A positive librarywas then used to isolate clones encoding the gene of interest using theprobe oligonucleotide and one of the primer pairs.

The oligonucleotide probes employed were as follows:

forward PCR primer (SEQ ID NO: 32) 5′-CCGACTTCTTGCAGGGCCGG-3′reverse PCR primer (SEQ ID NO: 33) 5′-GCAGCACGCAGCTGAGCGAG-3′hybridization probe (SEQ ID NO: 34)5′-AGCGAGTGGCTACAGGATGGGGTGTCCGGGCCC-3′

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 233-235 and a stop signal at nucleotide positions 2348-2350(FIG. 13, SEQ ID NO:13). The predicted polypeptide precursor is 705amino acids long, has a calculated molecular weight of approximately76,898 daltons and an estimated pI of approximately 6.08 Analysis of thefull-length PRO20040 sequence shown in FIG. 14 (SEQ ID NO:14) evidencesthe presence of a variety of important polypeptide domains as shown inFIG. 14, wherein the locations given for those important polypeptidedomains are approximate as described above. Clone DNA164625-2890 hasbeen deposited with ATCC on March 21, 2000 and is assigned ATCC depositno. PTA-1535.

An analysis of the Dayhoff database shows that PRO20040 has sequencesimilarity to an IL-17 receptor protein and PRO20040 is also designatedherein as IL-17RH2 as described in Example 20 of the presentapplication. Specifically, an analysis of the Dayhoff database (version35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis ofthe full-length sequence shown in FIG. 14 (SEQ ID NO:14), evidencedsequence identity between the PRO20040 amino acid sequence and thefollowing Dayhoff sequences: HSU58917_(—)1.

Example 7 Isolation of cDNA Clones Encoding a Human PRO9877

DNA119502-2789 was identified by applying a proprietary signal sequencefinding algorithm developed by Genentech, Inc. (South San Francisco,Calif.) upon ESTs as well as clustered and assembled EST fragments frompublic (e.g., GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals,Inc., Palo Alto, Calif.) databases. The signal sequence algorithmcomputes a secretion signal score based on the character of the DNAnucleotides surrounding the first and optionally the second methioninecodon(s) (ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals.

Use of the above described signal sequence algorithm allowedidentification of an EST cluster sequence from the LIFESEQ® database,designated herein as CLU42993. This EST cluster sequence was thencompared to a variety of expressed sequence tag (EST) databases whichincluded public EST databases (e.g., GenBank) and a proprietary EST DNAdatabase (LIFESEQ® Incyte Pharmaceuticals, Palo Alto, Calif.) toidentify existing homologies. The homology search was performed usingthe computer program BLAST or BLAST2 (Altshul et al., Methods inEnzymology 266:460-480 (1996)). Those comparisons resulting in a BLASTscore of 70 (or in some cases, 90) or greater that did not encode knownproteins were clustered and assembled into a consensus DNA sequence withthe program “phrap” (Phil Green, University of Washington, Seattle,Wash.). The consensus sequence obtained therefrom is herein designatedDNAFROM.

In light of an observed sequence homology between the DNAFROM sequenceand an EST sequence encompassed within clone no. 700536 from theLIFESEQ® database, clone no. 700536 was purchased and the cDNA insertwas obtained and sequenced. It was found herein that that cDNA insertencoded a full-length protein. The sequence of this cDNA insert is shownin FIG. 15 and is herein designated as DNA119502-2789.

Clone DNA119502-2789 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 106-108and ending at the stop codon at nucleotide positions 2107-2109 (FIG. 15;SEQ ID NO:15). The predicted polypeptide precursor is 667 amino acidslong (FIG. 16). The full-length PRO9877 protein shown in FIG. 16 has anestimated molecular weight of about 74,810 daltons and a pI of about9.55. Analysis of the full-length PRO9877 sequence shown in FIG. 16 (SEQID NO:16) evidences the presence of a variety of important polypeptidedomains as shown in FIG. 16, wherein the locations given for thoseimportant polypeptide domains are approximate as described above. CloneDNA119502-2789 has been deposited with ATCC on December 22, 1999 and isassigned ATCC deposit no. PTA-1082.

An analysis of the Dayhoff database shows that PRO9877 has sequencesimilarity to an IL-17 receptor protein and PRO9877 is also designatedherein as IL-17RH3. Specifically, an analysis of the Dayhoff database(version 35.45 SwissProt 35), using the ALIGN-2 sequence alignmentanalysis of the full-length sequence shown in FIG. 16 (SEQ ID NO:16),evidenced sequence identity between the PRO9877 amino acid sequence andthe following Dayhoff sequences: P_W61272, HSU58917_(—)1, P_W04185,P_W92409, GEN13979, P_W04184, P_W92408, MMU319931, P_W61271, andAF090114_(—)1.

Example 8 Isolation of cDNA Clones Encoding a Human PRO20026

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included a proprietary EST database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.). The search was performed using thecomputer program BLAST or BLAST2 [Altschul et al., Methods inEnzymology, 266:460-480 (1996)] as a comparison of the ECD proteinsequences to a 6 frame translation of the EST sequences. Thosecomparisons resulting in a BLAST score of 70 (or in some cases, 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.).

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described above. This consensus sequence is hereindesignated DNA149870. In some cases, the DNA149870 consensus sequencederives from an intermediate consensus DNA sequence which was extendedusing repeated cycles of BLAST and phrap to extend that intermediateconsensus sequence as far as possible using the sources of EST sequencesdiscussed above.

Based on the DNA149870 consensus sequence, flip cloning was performed.Oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO20026. Forwardand reverse PCR primers generally range from 20 to 30 nucleotides andare often designed to give a PCR product of about 100-1000 bp in length.The probe sequences are typically 40-55 bp in length. In some cases,additional oligonucleotides are synthesized when the consensus sequenceis greater than about 1-1.5 kbp. In order to screen several librariesfor a full-length clone, DNA from the libraries was screened by Flip PCRamplification, as per Schanke et al., BioTechniques, 16:414-416 (1994),with the PCR primer pair. A positive library was then used to isolateclones encoding the gene of interest using the probe oligonucleotide andone of the primer pairs.

PCR primers (forward and reverse) were synthesized: forward PCR primer:

(SEQ ID NO: 35) 5′-CGTTGTTTGTCAGTGGAGAGCAGGG-3′ reverse PCR primer(SEQ ID NO: 36) 5′-CAGGAACACCTGAGGCAGAAGCG-3′Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA149870 sequence which had thefollowing nucleotide sequence

hybridization probe (SEQ ID NO: 37)5′-CTATCTCCCTGCCAGGAGGCCGGAGTGGGGGAGGTCAGAC-3′

RNA for construction of the cDNA libraries was isolated from humantissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRKSD thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a full-length PRO20026 polypeptide(designated herein as DNA154095-2998 [FIG. 17, SEQ ID NO: 17]) and thederived protein sequence for that PRO20026 polypeptide.

The full length clone identified above contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 70-72 and a stop signal at nucleotide positions 2254-2256(FIG. 17, SEQ ID NO: 17). The predicted polypeptide precursor is 728amino acids long, has a calculated molecular weight of approximately81,310 daltons and an estimated pI of approximately 6.84. Analysis ofthe full-length PRO20026 sequence shown in FIG. 18 (SEQ ID NO: 18)evidences the presence of a variety of important polypeptide domains asshown in FIG. 18, wherein the locations given for those importantpolypeptide domains are approximate as described above. CloneDNA154095-2998 has been deposited with ATCC on October 10, 2000 and isassigned ATCC Deposit No. PTA-2591.

An analysis of the Dayhoff database shows that PRO20026 has sequencesimilarity to an IL-17 receptor protein and PRO2006 is also designatedherein as IL-17RH4. Specifically, an analysis of the Dayhoff database(version 35.45 SwissProt 35), using the ALIGN-2 sequence alignmentanalysis of the full-length sequence shown in FIG. 18 (SEQ ID NO: 18),evidenced sequence identity between the PRO20026 amino acid sequence andthe following Dayhoff sequences: T42695, P_W04185, P_W92409, P_W61272,NM_(—)014339_(—)1, HSU58917_(—)1, MMU31993_(—)1, GEN13979, P_W04184,P_W61271.

Example 9 Use of PRO as a Hybridization Probe

The following method describes use of a nucleotide sequence encoding PROas a hybridization probe.

DNA comprising the coding sequence of full-length or mature PRO asdisclosed herein is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of PRO) in humantissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled PRO-derived probe to the filters is performed in asolution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate,50 mM sodium phosphate, pH 6.8, 2× Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO can then be identified using standardtechniques known in the art.

Example 10 In Situ Hybridization

In situ hybridization is a powerful and versatile technique for thedetection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis and aid in chromosome mapping.

In situ hybridization was performed following an optimized version ofthe protocol by Lu and Gillett, Cell Vision, 1:169-176 (1994), usingPCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedded human tissues were sectioned, deparaffinized,deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., andfurther processed for in situ hybridization as described by Lu andGillett, supra.

A [³³-P] UTP-labeled antisense riboprobe was generated from a PCRproduct and hybridized at 55EC overnight. The slides were dipped inKodak NTB2 nuclear track emulsion and exposed for 4 weeks.

³³P-Riboprobe Synthesis

6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) werespeed vac dried. To each tube containing dried ³³P-UTP, the followingingredients were added:

2.0 μl 5× transcription buffer

1.0 μl DTT (100 mM)

2.0 μl NTP mix (2.5 mM: 10 μl; each of 10 mM GTP, CTP & ATP+10 μl H₂O)

1.0 μl UTP (50 μM)

1.0 μl Rnasin

1.0 μl DNA template (1 μg)

1.0 μl H₂O

1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

The tubes were incubated at 37° C. for one hour. 1.0 μl RQ1 DNase wereadded, followed by incubation at 37° C. for 15 minutes. 90 μl TE (10 mMTris pH 7.6/1 mM EDTA pH 8.0) were added, and mixture was pipetted ontoDE81 paper. The remaining solution was loaded in a Microcon-50ultrafiltration unit, and spun using program 10 (6 minutes). Thefiltration unit was inverted over a second tube and spun using program 2(3 minutes). After the final recovery spin, 100 μl TE were added. 1 μlof the final product was pipetted on DE81 paper and counted in 6 ml ofBiofluor II.

The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 μl of RNAMrk III were added to 3 μl of loading buffer. After heating on a 95ECheat block for three minutes, the gel was immediately placed on ice. Thewells of gel were flushed, the sample loaded, and run at 180-250 voltsfor 45 minutes. The gel was wrapped in saran wrap and exposed to XARfilm with an intensifying screen in −70EC freezer one hour to overnight.

³³P-Hybridization

A. Pretreatment of Frozen Sections

The slides were removed from the freezer, placed on aluminum trays andthawed at room temperature for 5 minutes. The trays were placed in 55°C. incubator for five minutes to reduce condensation.

The slides were fixed for 10 minutes in 4% paraformaldehyde on ice inthe fume hood, and washed in 0.5×SSC for 5 minutes, at room temperature(25 ml 20×SSC+975 ml SQ H₂O). After deproteination in 0.5 μg/mlproteinase K for 10 minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250ml prewarmed RNase-free RNase buffer), the sections were washed in0.5×SSC for 10 minutes at room temperature. The sections were dehydratedin 70%, 95%, 100% ethanol, 2 minutes each.

B. Pretreatment of Paraffin-Embedded Sections

The slides were deparaffinized, placed in SQ H₂O, and rinsed twice in2×SSC at room temperature, for 5 minutes each time. The sections weredeproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 mlRNase-free RNase buffer; 37° C., 15 minutes)−human embryo, or 8×proteinase K (100 μl in 250 ml RNase buffer, 37° C., 30minutes)−formalin tissues. Subsequent rinsing in 0.5×SSC and dehydrationwere performed as described above.

C. Prehybridization

The slides were laid out in a plastic box lined with Box buffer (4×SSC,50% formamide)−saturated filter paper. The tissue was covered with 50 μlof hybridization buffer (3.75g Dextran Sulfate+6 ml SQ H₂O), vortexedand heated in the microwave for 2 minutes with the cap loosened. Aftercooling on ice, 18.75 ml formamide, 3.75 ml 20×SSC and 9 ml SQ H₂O wereadded, the tissue was vortexed well, and incubated at 42° C. for 1-4hours.

D. Hybridization

1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heatedat 95° C. for 3 minutes. The slides were cooled on ice, and 48 μlhybridization buffer were added per slide. After vortexing, 50 μl ³³Pmix were added to 50 μl prehybridization on slide. The slides wereincubated overnight at 55° C.

E. Washes

Washing was done 2×10 minutes with 2×SSC, EDTA at room temperature (400ml 20×SSC+16 ml 0.25M EDTA, V_(f)=4 L), followed by RNaseA treatment at37° C. for 30 minutes (500 μl of 10 mg/ml in 250 ml RNase buffer=20μg/ml), The slides were washed 2×10 minutes with 2×SSC, EDTA at roomtemperature. The stringency wash conditions were as follows: 2 hours at55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, V_(f)=4 L).

F. Oligonucleotides

In situ analysis was performed on DNA59294-1381 disclosed herein. Theoligonucleotides employed for this analysis were derived from thenucleotide sequences disclosed herein and generally range from about 40to 55 nucleotides in length.

G. Results

In situ analysis was performed on DNA59294-1381 as disclosed herein. Theresults from this analysis is as follows.

DNA59294-1381 (PRO1031)

The expression of this IL17 homolog was evaluated in a panel consistingof normal adult and fetal tissues and tissues with inflammation,predominantly chronic lymphocytic inflammation. This panel is designedto specifically evaluate the expression pattern in immune mediatedinflammatory disease of novel proteins that modulate T lymphocytefunction (stimulatory or inhibitory). This protein when expressed as anIg-fusion protein was immunostimulatory in a dose dependent fashion inthe human mixed lymphocyte reaction (MLR); it caused a 285% and 147%increase above the baseline stimulation index when utilized at twodifferent concentrations (1.0% and 0.1% of a 560 nM stock) [see EXAMPLE25 below]. Summary: expression was restricted to muscle, certain typesof smooth muscle in the adult and in skeletal and smooth muscle in thehuman fetus. Expression in adult human was in smooth muscle of tubularorgans evaluated including colon and gall bladder. There was noexpression in the smooth muscle of vessels or bronchi. No adult humanskeletal muscle was evaluated. In fetal tissues there was moderate tohigh diffuse expression in skeletal muscle, in the axial skeleton andlimbs. There was weak expression in the smooth muscle of the intestinalwall but no expression in cardiac muscle. Adult human tissues withexpression include: Colon: there was low level diffuse expression in thesmooth muscle (tunica muscularis) in 5 specimens with chronicinflammatory bowel disease; Gall bladder: there was weak to low levelexpression in the smooth muscle of the gall bladder; Fetal human tissueswith expression: there was moderate diffuse expression in skeletalmuscle and weak to low expression in smooth muscle, there was noexpression in fetal heart or any other fetal organ including liver,spleen, CNS, kidney, gut, lung; Human tissues with no expression: lungwith chronic granulomatous inflammation and chronic bronchitis (5patients), peripheral nerve, prostate, heart, placenta, liver (diseasemulti block), brain (cerebrum and cerebellum), tonsil (reactivehyperplasia), peripheral lymph node, thymus.

Example 11 Expression of PRO in E. coli

This example illustrates preparation of an un glycosylated form of PROpolypeptides by recombinant expression in E. coll.

The DNA sequence encoding a PRO polypeptide is initially amplified usingselected PCR primers. The primers should contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector. A variety of expression vectors may be employed. Anexample of a suitable vector is pBR322 (derived from E. coli; seeBolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillinand tetracycline resistance. The vector is digested with restrictionenzyme and dephosphorylated. The PCR amplified sequences are thenligated into the vector. The vector will preferably include sequenceswhich encode for an antibiotic resistance gene, a trp promoter, apolyHis leader (including the first six STII codons, polyHis sequence,and enterokinase cleavage site), the PRO polypeptide coding region,lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

PRO polypeptides may be expressed in E. coli in a poly-His tagged form,using the following procedure. The DNA encoding a PRO polypeptide isinitially amplified using selected PCR primers. The primers will containrestriction enzyme sites which correspond to the restriction enzymesites on the selected expression vector, and other useful sequencesproviding for efficient and reliable translation initiation, rapidpurification on a metal chelation column, and proteolytic removal withenterokinase. The PCR-amplified, poly-His tagged sequences are thenligated into an expression vector, which is used to transform an E. colihost based on strain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts)clpP(laclq). Transformants are first grown in LB containing 50 mg/mlcarbenicillin at 30° C. with shaking until an O.D.600 of 3-5 is reached.Cultures are then diluted 50-100 fold into CRAP media (prepared bymixing 3.57 g (NH₄)₂SO₄, 0.71 g sodium citrate•2H2O, 1.07 g KCl, 5.36 gDifco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as wellas 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grownfor approximately 20-30 hours at 30° C. with shaking. Samples areremoved to verify expression by SDS-PAGE analysis, and the bulk cultureis centrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentrifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

The proteins are refolded by diluting the sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes are chosen so that the final protein concentration is between 50to 100 micrograms/ml. The refolding solution is stirred gently at 4° C.for 12-36 hours. The refolding reaction is quenched by the addition ofTFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution is filtered through a0.22 micron filter and acetonitrile is added to 2-10% finalconcentration. The refolded protein is chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance are analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein are pooled. Generally,the properly refolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded PRO polypeptide are pooled andthe acetonitrile removed using a gentle stream of nitrogen directed atthe solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14M sodium chloride and 4% mannitol by dialysis or by gel filtration usingG25 Superfine (Pharmacia) resins equilibrated in the formulation bufferand sterile filtered.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 12 Expression of PRO in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof PRO polypeptides by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO DNA is ligated into pRK5with selected restriction enzymes to allow insertion of the PRO DNAusing ligation methods such as described in Sambrook et al., supra. Theresulting vector is called pRK5-PRO.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μg pRK5-PRODNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappayaet al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl,0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added, dropwise, 500 μlof 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄, and a precipitateis allowed to form for 10 minutes at 25° C. The precipitate is suspendedand added to the 293 cells and allowed to settle for about four hours at37° C. The culture medium is aspirated off and 2 ml of 20% glycerol inPBS is added for 30 seconds. The 293 cells are then washed with serumfree medium, fresh medium is added and the cells are incubated for about5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of the PRO polypeptide. The cultures containing transfectedcells may undergo further incubation (in serum free medium) and themedium is tested in selected bioassays.

In an alternative technique, PRO may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. Thecells are first concentrated from the spinner flask by centrifugationand washed with PBS. The DNA-dextran precipitate is incubated on thecell pellet for four hours. The cells are treated with 20% glycerol for90 seconds, washed with tissue culture medium, and re-introduced intothe spinner flask containing tissue culture medium, 5 μg/ml bovineinsulin and 0.1 μg/ml bovine transferrin. After about four days, theconditioned media is centrifuged and filtered to remove cells anddebris. The sample containing the expressed PRO polypeptide can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

In another embodiment, PRO polypeptides can be expressed in CHO cells.The pRK5-PRO can be transfected into CHO cells using known reagents suchas CaPO₄ or DEAE-dextran. As described above, the cell cultures can beincubated, and the medium replaced with culture medium (alone) or mediumcontaining a radiolabel such as ³⁵S-methionine. After determining thepresence of the PRO polypeptide, the culture medium may be replaced withserum free medium. Preferably, the cultures are incubated for about 6days, and then the conditioned medium is harvested. The mediumcontaining the expressed PRO polypeptide can then be concentrated andpurified by any selected method.

Epitope-tagged PRO may also be expressed in host CHO cells. The PRO maybe subcloned out of the pRK5 vector. The subclone insert can undergo PCRto fuse in frame with a selected epitope tag such as a poly-His tag intoa Baculovirus expression vector. The poly-His tagged PRO insert can thenbe subcloned into a SV40 driven vector containing a selection markersuch as DHFR for selection of stable clones. Finally, the CHO cells canbe transfected (as described above) with the SV40 driven vector.Labeling may be performed, as described above, to verify expression. Theculture medium containing the expressed poly-His tagged PRO can then beconcentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

PRO polypeptides may also be expressed in CHO and/or COS cells by atransient expression procedure or in CHO cells by another stableexpression procedure.

Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g., extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domains,and/or as a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used in expression in CHOcells is as described in Lucas et al., Nucl. Acids Res., 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Qiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement intowater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3 L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH ie determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 jtm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins are purified using aNi-NTA column (Qiagen). Before purification, imidazole is added to theconditioned media to a concentration of 5 mM. The conditioned media ispumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5ml/min. at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydesalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 13 Expression of PRO in Yeast

The following method describes recombinant expression of PROpolypeptides in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO from the ADH2/GAPDH promoter. DNAencoding the PRO polypeptide and the promoter is inserted into suitablerestriction enzyme sites in the selected plasmid to direct intracellularexpression of the PRO polypeptide. For secretion, DNA encoding PRO canbe cloned into the selected plasmid, together with DNA encoding theADH2/GAPDH promoter, a native PRO signal peptide or other mammaliansignal peptide, or, for example, a yeast alpha-factor or invertasesecretory signal/leader sequence, and linker sequences (if needed) forexpression of PRO.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO polypeptides can subsequently be isolated and purifiedby removing the yeast cells from the fermentation medium bycentrifugation and then concentrating the medium using selectedcartridge filters. The concentrate containing the PRO polypeptide mayfurther be purified using selected column chromatography resins.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 14 Expression of PRO in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of PROpolypeptides in Baculovirus-infected insect cells.

The sequence coding for PRO is fused upstream of an epitope tagcontained within a Baculovirus expression vector. Such epitope tagsinclude poly-His tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding PRO or the desired portion of the coding sequence ofPRO such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-His tagged PRO can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaC1, 10%glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged PRO are pooled and dialyzedagainst loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PRO can beperformed using known chromatography techniques, including for instance,Protein A or protein G column chromatography.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 15 Preparation of Antibodies that Bind PRO

This example illustrates preparation of monoclonal antibodies which canspecifically bind PRO.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO polypeptides, fusion proteinscontaining PRO polypeptides, and cells expressing recombinant PROpolypeptides on the cell surface. Selection of the immunogen can be madeby the skilled artisan without undue experimentation.

Mice, such as BALB/c, are immunized with the PRO immunogen emulsified incomplete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstPRO. Determination of “positive” hybridoma cells secreting the desiredmonoclonal antibodies against PRO is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic BALB/c mice to produce ascites containing the anti-PROmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 16 Purification of PRO Polypeptides Using Specific Antibodies

Native or recombinant PRO polypeptides may be purified by a variety ofstandard techniques in the art of protein purification. For example,pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide ispurified by immunoaffinity chromatography using antibodies specific forthe PRO polypeptide of interest. In general, an immunoaffinity column isconstructed by covalently coupling the anti-PRO polypeptide antibody toan activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PROpolypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A soluble PRO polypeptide-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of PRO polypeptide (e.g., high ionicstrength buffers in the presence of detergent). Then, the column iseluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

Example 17 Drug Screening

This invention is particularly useful for screening compounds by usingPRO polypeptides or binding fragment thereof in any of a variety of drugscreening techniques. The PRO polypeptide or fragment employed in such atest may either be free in solution, affixed to a solid support, borneon a cell surface, or located intracellularly. One method of drugscreening utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO polypeptide-associated diseaseor disorder. These methods comprise contacting such an agent with an PROpolypeptide or fragment thereof and assaying (i) for the presence of acomplex between the agent and the PRO polypeptide or fragment, or (ii)for the presence of a complex between the PRO polypeptide or fragmentand the cell, by methods well known in the art. In such competitivebinding assays, the PRO polypeptide or fragment is typically labeled.After suitable incubation, free PRO polypeptide or fragment is separatedfrom that present in bound form, and the amount of free or uncomplexedlabel is a measure of the ability of the particular agent to bind to PROpolypeptide or to interfere with the PRO polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO polypeptide, the peptide test compounds arereacted with PRO polypeptide and washed. Bound PRO polypeptide isdetected by methods well known in the art. Purified PRO polypeptide canalso be coated directly onto plates for use in the aforementioned drugscreening techniques. In addition, non-neutralizing antibodies can beused to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

Example 18 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO polypeptide) orof small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (c. f, Hodgson, Bio/Technology, 9: 19-21 (1991)).

In one approach, the three-dimensional structure of the PRO polypeptide,or of an PRO polypeptide-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry, 31:7796-7801(1992) or which act as inhibitors, agonists, or antagonists of nativepeptides as shown by Athauda et al., J. Biochem., 113:742-746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

Example 19 Differential Tissue Expression Distribution

Oligonucleotide probes were constructed from the PRO1031, PRO1122,PRO21175, PRO10272, PRO20110, PRO5801, PRO20040, PRO9877, and PRO20026polypeptide-encoding nucleotide sequences shown in the accompanyingfigures for use in quantitative PCR amplification reactions. Theoligonucleotide probes were chosen so as to give an approximately200-600 base pair amplified fragment from the 3′ end of its associatedtemplate in a standard PCR reaction. The oligonucleotide probes wereemployed in standard quantitative PCR amplification reactions with cDNAlibraries isolated from different human adult and/or fetal tissuesources and analyzed by agarose gel electrophoresis so as to obtain aquantitative determination of the level of expression of thepolypeptide-encoding nucleic acids in the various tissues tested.Knowledge of the expression pattern or the differential expression ofthe polypeptide-encoding nucleic acid in various different human tissuetypes provides a diagnostic marker useful for tissue typing, with orwithout other tissue-specific markers, for determining the primarytissue source of a metastatic tumor, and the like. These assays providedthe following results:

DNA Molecule Tissues w/Significant Expression Tissues w/o SignificantExpression DNA59294-1381 highly expressed in mammary gland, weaklyexpressed in intestine, bone trachea, testis and spine marrow, lung,kidney and thymus; no expression in pancreas, liver, brain or spleenDNA62377-1381-1 strongly expressed in testis, spleen, weakly expressedin intestine, fetal thymus, and trachea brain, mammary, uterus, colon,lung, placenta and stomach; no expression in muscle, pancreas, liver,spine, brain and fetal liver DNA173894-2974 highly expressed in muscle,spine not expressed in intestine, mammary and brain gland, marrow,uterus, trachea, colon, salivary gland, lung, pancreas, liver, prostate,adrenal, kidney, thymus, placenta, heart, stomach and spleenDNA147531-2821 expressed at low levels in brain, no expression in heart,liver, colon, kidney, lung, prostate, testis, spinal marrow, intestine,spleen, muscle, chord, adrenal gland and trachea stomach, uterus,placenta, thymus, muscle, uterus, placenta, pancreas, salivary gland,and mammary gland DNA166819 highly expressed in testis, kidney notexpressed in intestine, mammary thymus, and stomach gland, marrow,uterus, trachea, colon, salivary gland, lung, muscle, pancreas, liver,prostate, adrenal gland, placenta heart, spine, brain, and spleenDNA115291-2681 highly expressed in the kidney; not expressed in heart,bone marrow, significant expression in liver and spleen and placentaperipheral organs such as colon, small intestine, prostate, testis,pancreas and uterus DNA164625-2890 highly expressed in prostate;expressed in weakly expressed in heart, cartilage, kidney, spine,placenta, liver, lung, colon, colon tumor, substantia nigra and spleen,uterus, dendrocyte and and macrophage; it is not expressed inhippocampus, intestine, mammary gland, lymphoblasts bone marrow, testis,muscle, stomach and thymus DNA119502-2789 strongly expressed in mammarynot expressed in muscle, liver, and gland, placenta and prostate;expressed heart; weakly expressed in marrow, in intestine, colon, lung,kidney, thymus, uterus, testis and brain stomach, spine and spleenDNA154095-2998 strongly expressed in fetal brain; negligible expressionin mammary significant expression in uterus and gland, bone marrow,trachea, colon, testis; expressed in prostate, esophagus lung, muscle,pancreas, liver, and esophagial tumors, normal adrenal gland, thymus,placenta, stomach and stomach tumor, kidney but heart, brain and spleen,rectum; not expressed higher in kidney tumor, lung expressed in livertumor tumor, and rectal tumor

Example 20 Identification of Receptor/Ligand Interactions—Overview ofScreening Assay of PRO Polypeptides for Identification ofReceptor/Ligand Interactions

In this assay, various PRO polypeptides are tested for ability to bindto a panel of potential receptor or ligand molecules for the purpose ofidentifying receptor/ligand interactions. The identification of a ligandfor a known receptor, a receptor for a known ligand or a novelreceptor/ligand pair is useful for a variety of indications including,for example, targeting bioactive molecules (linked to the ligand orreceptor) to a cell known to express the receptor or ligand, use of thereceptor or ligand as a reagent to detect the presence of the ligand orreceptor in a composition suspected of containing the same, wherein thecomposition may comprise cells suspected of expressing the ligand orreceptor, modulating the growth of or another biological orimmunological activity of a cell known to express or respond to thereceptor or ligand, modulating the immune response of cells or towardcells that express the receptor or ligand, allowing the preparation ofagonists, antagonists and/or antibodies directed against the receptor orligand which will modulate the growth of or a biological orimmunological activity of a cell expressing the receptor or ligand, andvarious other indications which will be readily apparent to theordinarily skilled artisan.

In general, the assay is performed as follows. A PRO polypeptide of thepresent invention suspected of being a ligand for a receptor isexpressed as a fusion protein containing the Fc domain of human IgG (animmunoadhesin). Receptor-ligand binding is detected by allowinginteraction of the immunoadhesin polypeptide with cells (e.g. Cos cells)expressing candidate PRO polypeptide receptors and visualization ofbound immunoadhesin with fluorescent reagents directed toward the Fcfusion domain and examination by microscope. Cells expressing candidatereceptors are produced by transient transfection, in parallel, ofdefined subsets of a library of cDNA expression vectors encoding PROpolypeptides that may function as receptor molecules. Cells are thenincubated for 1 hour in the presence of the PRO polypeptideimmunoadhesin being tested for possible receptor binding. The cells arethen washed and fixed with paraformaldehyde. The cells are thenincubated with fluorescent conjugated antibody directed against the Fcportion of the PRO polypeptide immunoadhesin (e.g. FITC conjugated goatanti-human-Fc antibody). The cells are then washed again and examined bymicroscope. A positive interaction is judged by the presence offluorescent labeling of cells transfected with cDNA encoding aparticular PRO polypeptide receptor or pool of receptors and an absenceof similar fluorescent labeling of similarly prepared cells that havebeen transfected with other cDNA or pools of cDNA. If a defined pool ofcDNA expression vectors is judged to be positive for interaction with aPRO polypeptide immunoadhesin, the individual cDNA species that comprisethe pool are tested individually (the pool is “broken down”) todetermine the specific cDNA that encodes a receptor able to interactwith the PRO polypeptide immunoadhesin.

In another embodiment of this assay, an epitope-tagged potential ligandPRO polypeptide (e.g., 8 histidine “His” tag) is allowed to interactwith a panel of potential receptor PRO polypeptide molecules that havebeen expressed as fusions with the Fc domain of human IgG(immunoadhesins). Following a 1 hour co-incubation with the epitopetagged PRO polypeptide, the candidate receptors are eachimmunoprecipitated with protein A beads and the beads are washed.Potential ligand interaction is determined by Western blot analysis ofthe immunoprecipitated complexes with antibody directed towards theepitope tag. An interaction is judged to occur if a band of theanticipated molecular weight of the epitope tagged protein is observedin the Western blot analysis with a candidate receptor, but is notobserved to occur with the other members of the panel of potentialreceptors.

Using the above described assays, the following receptor/ligandinteractions have been herein identified:

-   (1) PRO1031 (designated herein as human IL-17B ligand) binds to    PRO5801 (designated herein as human IL-17RH1 receptor).-   (2) PRO10272 (designated herein as human IL-17E ligand) binds to    PRO5801 (designated herein as human IL-17RH1 receptor).-   (3) PRO20110 (designated herein as human IL-17F ligand) binds to the    human IL-17 receptor (IL-17R) [(Yao et al., Cytokine, 9(11):794-800    (1997); also herein designated as PRO1] and to PRO20040 (designated    herein as human IL-17RH2 receptor).-   (4) PRO1031 (IL-17B ligand) and PRO1122 (IL-17C ligand) do not bind    to the human IL-17 receptor (Li et al., Proc. Natl. Acad. Sci.    (USA), 97(2):773-778 (2000)).

Example 21 Human IL-17 Receptor (IL-17R; Designated PRO1) Binding withNovel Ligands IL-17B (Designated PRO1031) and IL-17C (DesignatedPRO1122)

A. Cloning of the ECD of Human IL-17 Receptor (Designated IL-17R;Designated herein as PRO1):

The ECD of human IL-17 receptor (IL-17R) [Yao et al., Cytokine9(11):794-800 (1997)] was cloned in order to study the ligand/receptorinteractions of the novel IL-17 homolog polypeptides IL-17B and IL-17C.Two oligonucleotide primers were designed at the 5′ and 3′ ends of thehuman IL-17R ECD based on the published sequence. [Yao et al., Cytokine,9:794 (1997)]. The two probes had the following sequences:

primer 1: (SEQ ID NO: 38) 5′-CTG TAC CTC GAG GGT GCA GAG-3′ primer 2:(SEQ ID NO: 39) 5′-CCC AAG CTT GGG TCA ATG ATG ATG ATG ATG ATGATG ATG CCA CAG GGG CAT GTA GTC C-3′

The above primers were used in PCR reactions to amplify the full-lengthcDNA from a human testis cDNA library with Pfu Turbo DNA polymerase(Promega). A C-terminal His tag was introduced by PCR through theaddition of nucleotides encoding eight histidines to the 3′ end primer.The PCR product was then subcloned into an expression plasmid vectorpRK5B. Sequence analysis confirmed that the insert contains a DNAfragment encoding the extracellular domain (1-320 amino acids) of thepublished hIL-17 receptor.

B. Immunoprecipitation of the IL-17R ECD:

The differential activity of IL-17 when compared to IL-17B (PRO1031;SEQID NO:2) and IL-17C (PRO1122; SEQ ID NO:4) (see Examples 28 through 30of the present application) suggested that they might bind and activatedifferent cell surface receptors. In order to test whether IL-17B(PRO1031) or IL-17C (PRO1122) directly bind to the receptor, anexpression plasmid containing the IL-17R (PRO1)(C-terminal His-tagged)was transfected into 293 cells using SuperFect transfection reagent(Qiagen). Metabolic labeling of 293 cells was performed 16 hours aftertransfection using 50 mCi/ml [³⁵S]-Cys/Met mixture for 6 hours.Conditioned medium was collected and concentrated (Centricon-10,Amicon). To examine the expression of the IL-17R ECD, Ni-NTA beads(Qiagen) were used to affinity precipitate the His-tagged IL-17R ECDfrom the conditioned medium.

The conditioned medium was diluted in RIPA buffer (1% NP40, 0.5% sodiumdeoxycholate, 0.1% SDS in PBS) and was incubated with IL-17 and the Fcfusion proteins overnight at 4° C. Protein A-agarose beads (Pierce) wereadded to precipitate the Fc fusion proteins. The precipitates werewashed three times to precipitate the Fc fusion proteins. Theprecipitates were washed three times in RIPA buffer, denatured in SDSsample buffer, and electrophoresed on NuPAGE 4-12% Bis-Tris gels(Novex). For IL-17 immunoprecipitation, anti-IL-17 antibody (R&DSystems) was added. In a competitive binding experiment,immunoprecipitation of IL-17R ECD by IL-17 is performed in the presenceof a 5-fold molar excess of IL-17B.His, IL-17C.His and control histagged protein.

The IL-17R ECD migrated as a 60 kDa band when purified via its histidinetag (FIG. 29A.), lane 1). Furthermore, the IL-17R ECD also precipitatedin combination with IL-17 (lane 3). However, both IL-17B and IL-17Cfailed to compete for the binding of IL-17 for the labeled IL-17receptor ECD (FIG. 29B.), lane 15 and 16).

Example 22 Novel Human IL-17 Receptor (IL-17RH1) (Designated PRO5801)Binding with Human IL-17 and Novel Ligands IL-17B (Designated PRO1031),IL-17C (Designated PRO1122, and IL-17E (Designated PRO10272); Inductionof NF-κB Activity and IL-8 Production by IL-17E A. Isolation of IL-17E(PRO10272) and Construction of Expression Vectors:

IL-17E (DNA147531-2821; SEQ ID NO:5) and IL-17RH1 (DNA115291-2681; SEQID NO:11) cDNA clones were isolated from a human cDNA library andsequenced in their entirety as described in EXAMPLE 3 and EXAMPLE 5,respectively. Fc fusion proteins (immunoadhesions) were prepared byfusion of the entire open reading frames of IL-17, IL-17B (PRO1031),IL-17C (PRO1122), and IL-17E (PRO10272) in frame with the Fc region ofhuman IgG1 in the eukaryotic expression vector pRK5tkNEO and thebaculovirus vector pHIF, a derivative of pVL1393 purchased fromPharmingen. Fusion proteins were transiently expressed in human 293cells or Sf9 insect cells and purified over a Protein A column. Theextracellular domain of the IL-17RH1 receptor (PRO5801) was alsoexpressed as a C-terminal 8xHis-tag fusion in baculovirus and purifiedby nickel affinity column. IL-17E (PRO10272) was also expressed as a8×His-tag fusion in E. coli and was purified and refolded. Theidentities of the purified proteins were verified by N-terminal sequenceanalysis.

B. Western Blot, Northern Blot and Taqman™ Analysis:

Western blot analysis of binding of IL-17E (PRO10272) to IL-17RH1(PRO5801) was performed essentially as described by Xie et al.,Cytokine, 11(10):729-735 (1999) and Xie et al., J. Biol. Chem., 275(40):31335-31339 (2000). For Northern blot analysis, multiple tissue Northernblots (Clontech) were probed with a ³²P-labeled probe of random primedIL-17RH1 cDNA according to manufacturer's recommendations and exposed toX-omat (Kodak) for 72 hours. For quantitative PCR analysis (Taqman™),total mRNA from human tissues (50 ng) was analyzed as recommended(Perkin Elmer) with primers based on the coding sequence of IL-17RH1.

C. FACS Analysis:

Human 293 cells were transiently co-transfected with expression vectorsfor green fluorescent protein (GFP), and IL-17RH1 (PRO5801) or IL-17R(designated PROD as indicated. After 24 hours, cells were incubated withFc tagged ligand as indicated and binding was revealed with PEconjugated anti-human Fc antibody. FACS curves show PE staining withinthe co-transfected GFP positive cell population. (FIG. 32A).

D. NF-κB, and IL-8 Assays and Western Blot Analysis:

Luciferase reporter assays were conducted essentially as described byGurney et al., Curr Biol., 9(4)):215-218 (1999). Briefly, 293 or TK-10cells (2×10⁵) were transfected by Effectine (Qiagen) transfection with0.5 Φg of the firefly luciferase reporter plasmid pGL3-ELAM.tk and 0.05Φg of the Renilla luciferase reporter plasmid as internal transfectioncontrol as well as IL-17E expression plasmid (0.1 Φg) and carrierplasmid pRK5D to maintain constant DNA between transfections. After 24hours cells were harvested and luciferase activity assayed asrecommended (Pharmacia). IL-8 ELISA were performed according tomanufacturer's instructions R&D) Systems). (FIG. 33)

E. Results and Discussion:

As described supra, novel members of IL-17 family have been identifiedand characterized, designated herein as IL-17B (PRO1031), IL-17C(PRO1122), IL-17D (PRO21175), and IL-17E (PRO10272). Four members of theIL-17 family: IL-17, IL-17B, IL-17C and IL-17E share greatest similarityin the C-terminal portion of the molecule with 20-30% amino acidsequence identity and strict conservation of four cysteines. Additionalcysteines that may be functionally conserved are present withdifferences in position. In contrast, there is little conservationapparent in the N-terminal 80 residues. The alignment of the IL-17family members [IL-17 (SEQ ID NO:40); IL-17B (PRO1031; SEQ ID NO:2);IL-17C (PRO1122, SEQ ID NO:4); and IL-17E (PRO10272, SEQ ID NO:6)] isdemonstrated in FIG. 30. The predicted signal sequences are underlined.Conserved cysteines are indicated by bullet, and potential N-linkedglycosylation sites are boxed.

IL-17E mRNA was not detected by Northern blot analysis. However, IL-17Ewas detected at very low levels in several tissues including brain,kidney, lung, prostate, testis, spinal chord, adrenal gland and tracheaby RT-PCR using primers designed to distinguish spliced mRNA fromgenomic DNA. The results of RT-PCR analysis of IL-17E (PRO10272)expression is shown in FIG. 23. As described above, RNA from theindicated tissues was subjected to RT-PCR with primers that weredesigned to amplify the entire coding sequence of IL-17E. The PCRproduct was resolved by agarose gel electrophoresis, transferred tonylon membrane and probed with a ³²P labeled IL-17E cDNA probe.

Applicants have demonstrated that IL-17B (PRO1031) and IL-17C (PRO1122)do not bind to the human IL-17 receptor (designated herein PRO1)(seeEXAMPLE 21). A novel IL-17 receptor (designated herein as IL-17RH1;PRO5801) has been herein identified and characterized. IL-17RH1(DNA115291-2681; SEQ ID NO:11) cDNA clones were isolated from a humancDNA library and sequenced in their entirety as described in EXAMPLE 5.IL-17RH1 mRNA expression was examined by Northern blot analysis as shownin FIG. 31A and quantitative PCR as shown in FIG. 31B. Highest levels ofexpression of IL-17RH1 (PRO5801) were observed in kidney, withsignificant expression also observed in liver, and other peripheralorgans such as colon, small intestine, prostate, testis, pancreas anduterus.

Binding studies were conducted to determine whether this new molecule(designated IL-17RH1; PRO5801) serves as a receptor for other members ofIL-17 family. Human 293 kidney cells transfected with an expressionvector for IL-17RH1 were shown to bind to IL-17E-Fc fusion protein(immunoadhesin), but do not show significant binding of human IL-17 (asshown in FIG. 32A). IL-17E immunoadhesin binding to IL-17RH1 expressingcells could be completely inhibited by competition with His epitopetagged IL-17E. In comparison, cells transfected with expression vectorfor IL-17R bind IL-17 immunoadhesin but not IL-17E. To examine whetherthere was direct interaction with members of the IL-17 family, ligandbinding studies were conducted with epitope tagged extracellular domainof the IL-17RH1 receptor. As shown in FIG. 32B, this novel receptorexhibits strong binding of IL-17E-Fc, and weak binding to IL-17B-Fc butdoes not bind IL-17-Fc or IL-17C-Fc.

IL-17 has been observed to induce NF-κB activity (Jovanovic et al.,supra). A study was done to determine whether IL-17E (PRO10272) wouldalso induce activation of a NF-κB responsive luciferase reporter gene intwo human renal cell carcinoma cell lines, 293 and TK-10 cells (both ofthese cell lines were found to express endogenous IL-17RH1 mRNA). Theresults of these studies are shown in FIG. 33A. Transfection ofexpression vector for IL-17E markedly induced luciferase activity. Theluciferase activity was induced in a dose dependent manner, and was ofsimilar magnitude to that observed by the overexpression of the TNFreceptor superfamily member GITR (see FIG. 33B), previously shown to bea potent inducer of NF-κB activity (Gurney et al., supra). NF-κB isthought to mediate a proinflammatory signal, suggesting that IL-17E mayhave proinflammatory action. To examine this possibility, the productionof IL-8, a proinflammatory chemokine induced by IL-17, was examined. Asshown in FIG. 34, IL-17E (PRO10272) induced activation of IL-8 in TK-10cells.

In summary, IL-17RH1 (PRO5801) is the second receptor identified whichbinds to members of the IL-17 family. The IL-17 receptor family is quiteunrelated to other proteins. However, comparison of the two receptorsdoes reveal conservation of many cysteines within the extracellulardomain, suggesting they share similar structure. There are conservedelements within the intracellular domain as well, suggesting that thesereceptors likely engage similar intracellular machinery. This issupported by the observation that like IL-17, IL-17E signals activationof NK-6B. The regions of conservation within the intracellular domain donot bear obvious similarity to other receptor families known to activateNF-κB, the IL1/Toll and TNF receptor families.

IL-17E induces production of IL-8, a proinflammatory molecule that hasalso been observed to be induced by IL-17, suggesting the biologicalactivities of these two cytokines may be similar. The IL-17 receptor hasa very broad expression pattern, in contrast to the somewhat morerestricted mRNA expression pattern of IL-17RH1 (PRO5801) (see FIG. 31).If these molecules mediate generally analogous proinflammatoryresponses, a key consideration in understanding the function of thedifferent members of the expanding IL-17 cytokine family will be theexpression patterns and regulation of the cognate receptors.

FIGS. 25 through 28 show the relative tissue expression distribution forthe novel IL-17 receptor homologs identified herein as IL-17RH1(PRO5801; SEQ ID NO:12), IL-17RH2 (PRO20040; SEQ ID NO:14), IL-17RH3(PRO9877; SEQ ID NO:16) and IL-17RH4 (PRO20026; SEQ ID NO:18),respectively.

In summary, FIG. 35 depicts the IL-17 family of cytokines complexpattern of overlapping receptor-ligand specificities. As shown, ligandsIL-17C and IL-17D appear to have specificity for a differentinterleukin-17 receptor other than IL-17R, IL-17RH1 or IL-17RH2. Inaddition, FIGS. 20 through 28 and FIG. 31 demonstrate the relativetissue expression distribution for the novel IL-17 homologs and IL-17receptors identified herein.

Example 23 Induction of c-fos in Endothelial Cells (ASSAY #34)

This assay is designed to determine whether PRO polypeptides show theability to induce c-fos in endothelial cells. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of conditions or disorders where angiogenesiswould be beneficial including, for example, wound healing, and the like(as would agonists of these PRO polypeptides). Antagonists of the PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of cancerous tumors.

Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) ingrowth media (50% Ham's F12 w/o GHT: low glucose, and 50% DMEM withoutglycine: with NaHCO₃, 1% glutamine, 10 mM HEPES, 10% FBS, 10 ng/ml bFGF)are plated on 96-well microtiter plates at a cell density of 1×10⁴cells/well. The day after plating, the cells are starved by removing thegrowth media and treating the cells with 100 μl/well test samples andcontrols (positive control: growth media; negative control: 10 mM HEPES,140 mM NaCl, 4% (w/v) mannitol, pH 6.8). The cells are incubated for 30minutes at 37° C., in 5% CO₂. The samples are removed, and the firstpart of the bDNA kit protocol (Chiron Diagnostics, cat. #6005-037) isfollowed, where each capitalized reagent/buffer listed below isavailable from the kit.

Briefly, the amounts of the TM Lysis Buffer and Probes needed for thetests are calculated based on information provided by the manufacturer.The appropriate amounts of thawed Probes are added to the TM LysisBuffer. The Capture Hybridization Buffer is warmed to room temperature.The bDNA strips are set up in the metal strip holders, and 100 μl ofCapture Hybridization Buffer are added to each b-DNA well needed,followed by incubation for at least 30 minutes. The test plates with thecells are removed from the incubator, and the media are gently removedusing the vacuum manifold. 100 μl of Lysis Hybridization Buffer withProbes are quickly pipetted into each well of the microtiter plates. Theplates are then incubated at 55° C. for 15 minutes. Upon removal fromthe incubator, the plates are placed on the vortex mixer with themicrotiter adapter head and vortex on the #2 setting for one minute. 80μl of the lysate are removed and added to the bDNA wells containing theCapture Hybridization Buffer, and pipetted up and down to mix. Theplates are incubated at 53° C. for at least 16 hours.

On the next day, the second part of the bDNA kit protocol is followed.Specifically, the plates are removed from the incubator and placed onthe bench to cool for 10 minutes. The volumes of additions needed arecalculated based upon information provided by the manufacturer. AnAmplifier Working Solution is prepared by making a 1:100 dilution of theAmplifier Concentrate (20 fm/μl) in AL Hybridization Buffer. Thehybridization mixture is removed from the plates and washed twice withWash A. 50 μl of Amplifier Working Solution are added to each well andthe wells are incubated at 53E C for 30 minutes. The plates are thenremoved from the incubator and allowed to cool for 10 minutes. The LabelProbe Working Solution is prepared by making a 1:100 dilution of LabelConcentrate (40 pmoles/μl) in AL Hybridization Buffer. After the10-minute cool-down period, the amplifier hybridization mixture isremoved and the plates are washed twice with Wash A. 50 μl of LabelProbe Working Solution are added to each well and the wells areincubated at 53° C. for 15 minutes. After cooling for 10 minutes, theSubstrate is warmed to room temperature. Upon addition of 3 μl ofSubstrate Enhancer to each ml of Substrate needed for the assay, theplates are allowed to cool for 10 minutes, the label hybridizationmixture is removed, and the plates are washed twice with Wash A andthree times with Wash D. 50 μl of the Substrate Solution with Enhancerare added to each well. The plates are incubated for 30 minutes at 37°C. and RLU is read in an appropriate luminometer.

The replicates are averaged and the coefficient of variation isdetermined. The measure of activity of the fold increase over thenegative control (HEPES buffer described above) value is indicated bychemiluminescence units (RLU). Samples that show an at least two-foldvalue over the negative control value are considered positive.

PRO1031 assayed “positive” as shown below:

ASSAY #1 Negative control = 1.0 RLU Positive control = 10.96 RLU PRO1031at 0.056 nM = 2.22 RLU ASSAY #2 Negative control = 1.0 RLU Positivecontrol = 10.96 RLU PRO1031 at 0.56 nM = 2.01 RLU

Example 24 Skin Vascular Permeability Assay (ASSAY #64)

This assay shows that certain PRO polypeptides stimulate an immuneresponse and induce inflammation by inducing mononuclear cell,eosinophil and PMN infiltration at the site of injection of the animal.This skin vascular permeability assay is conducted as follows. Hairlessguinea pigs weighing 350 grams or more are anesthetized with ketamine(75-80 mg/Kg) and 5 mg/Kg Xylazine intramuscularly (IM). A sample ofpurified PRO polypeptide or a conditioned media test sample is injectedintradermally onto the backs of the test animals with 100 μL perinjection site. It is possible to have about 10-30, preferably about16-24, injection sites per animal. One mL of Evans blue dye (1% inphysiologic buffered saline) is injected intracardially. Blemishes atthe injection sites are then measured (mm diameter) at 1 hr, 6 hrs and24 hrs post injection. Animals were sacrificed at 6 hrs after injection.Each skin injection site is biopsied and fixed in paraformaldehyde. Theskins are then prepared for histopathalogic evaluation. Each site isevaluated for inflammatory cell infiltration into the skin. Sites withvisible inflammatory cell inflammation are scored as positive.Inflammatory cells may be neutrophilic, eosinophilic, monocytic orlymphocytic.

At least a minimal perivascular infiltrate at the injection site isscored as positive, no infiltrate at the site of injection is scored asnegative. PRO1031 gave positive results at time interval 24 hours inthis assay.

Example 25 Stimulatory Activity in Mixed Lymphocyte Reaction (MLR)(ASSAY #24)

This example shows that the polypeptides of the invention are active asstimulators of the proliferation of T-lymphocytes. Compounds whichstimulate proliferation of lymphocytes are useful therapeutically whereenhancement of an immune response is beneficial. A therapeutic agent mayalso take the form of antagonists of the PRO polypeptides of theinvention, for example, murine-human chimeric, humanized or humanantibodies against the polypeptide, which would be expected to inhibitT-lymphocyte proliferation.

The basic protocol for this assay is described in Current Protocols inImmunology, unit 3.12; edited by J. E. Coligan, A. M. Kruisbeek, D. H.Marglies, E. M. Shevach, W. Strober, National Institutes of Health,Published by John Wiley & Sons, Inc.

More specifically, in one assay variant, peripheral blood mononuclearcells (PBMC) are isolated from mammalian individuals, for example ahuman volunteer, by leukopheresis (one donor will supply stimulatorPBMCs, the other donor will supply responder PBMCs). If desired, thecells are frozen in fetal bovine serum and DMSO after isolation. Frozencells may be thawed overnight in assay media (37° C., 5% CO₂)and thenwashed and resuspended to 3×10⁶ cells/ml of assay media (RPMI; 10% fetalbovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%non-essential amino acids, 1% pyruvate). The stimulator PBMCs areprepared by irradiating the cells (about 3000 Rads). The assay isprepared by plating in triplicate wells a mixture of: 100:1 of testsample diluted to 1% or to 0.1%; 50:1 of irradiated stimulator cells and50:1 of responder PBMC cells. 100 microliters of cell culture media or100 microliter of CD4-IgG is used as the control. The wells are thenincubated at 37° C., 5% CO₂ for 4 days. On day 5 and each well is pulsedwith tritiated thymidine (1.0 mCi/well; Amersham). After 6 hours thecells are washed 3 times and then the uptake of the label is evaluated.

In another variant of this assay, PBMCs are isolated from the spleens ofBALB/c mice and C57B6 mice. The cells are teased from freshly harvestedspleens in assay media (RPMI;10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above. Positive increases overcontrol are considered positive with increases of greater than or equalto 180% being preferred. However, any value greater than controlindicates a stimulatory effect for the test protein. The results of thisassay for compounds of the invention are shown below:

PRO PRO Concentration Percent Increase Over Control PRO10272 0.84 nM201.5

Example 26 Stimulation of Peripheral Blood Mononuclear Cells (PBMCs) orCD4⁺ Cells with Anti CD3 and PRO Protein (ASSAY #99)

This assay shows that one or more of the PRO polypeptides are active asenhancers of the stimulation of PBMCs or CD4⁺ cells. CD4⁺ cells areenriched by negative selection using MACs beads after LSM separation.The ability of the PRO polypeptide to replace anti-CD28 is examined todetermine the stimulatory effect.

Anti-CD3 and anti-CD28 are known to stimulate PBMCs. The basic protocolfor the isolation of PBMCs used in this assay is described in CurrentProtocols in Immunology, unit 3.1.2; edited by J. E. Coligan, A. M.Kruisbeek, D. H. Marglies, E. M. Shevach, W. Strober, NationalInstitutes of Health, Published by John Wiley & Sons, Inc. (1993).

More specifically, in one assay variant, peripheral blood mononuclearcells (PBMC) are isolated from mammalian individuals, for example ahuman volunteer, by leukopheresis. If desired, the cells are enrichedfor CD4⁻ cells, then frozen in 90% fetal bovine serum and 10% DMSO afterisolation. Frozen cells may be thawed overnight in assay media (37EC, 5%CO_(T)) and then washed and resuspended to 0.5×10⁶ cells/ml of assaymedia (RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1%glutamine, 1% HEPES, 1% non-essential amino acids, 1% pyruvate).

The assay is prepared by plating in triplicate wells a mixture of: 200Φl of cells after the overnight coat of anti-CD3 and PRO protein.

50 μl of anti-CD3 (50 ng/ml, Amac 0178) and 50 μl of 1% of the PROprotein are coated on a 96 well plate in PBS 4° C. overnight. 50 μlHu-IgG is used as the control in place of the PRO protein. The wells arethen incubated at 37° C., 5% CO₂ for about 3 days. On day 4, each wellis pulsed with tritiated thymidine (1.0 mC/well; Amersham). After 6hours the cells are harvested and then the uptake of the label isevaluated.

A result which indicates a stimulatory effect (i.e., ³[H]-thymidineincorporation) greater than 200% of the control is considered to bepositive stimulatory result.

In another variant of this assay, PBMCs or CD4⁺ splenocytes are isolatedfrom the spleens of BALB/c mice. The cells are teased from freshlyharvested spleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia. CD4⁺ cells are enriched by negative selection using beads, washedin media and resuspended the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above. Results are shown below:

PRO concentration stimulation (+)/inhibition (−) PRO1031 5.6 nM (+) 285%above baseline stimulation index PRO1031 0.56 nM (+) 147% above baselinestimulation index

Example 27 Generating IL-17B and IL-17C Fc/His Fusion Proteins

The coding sequences of IL17B and IL17C were amplified by PCR andsubcloned into the EcoRI and Smal sites of pBPH.His.c to generate aC-terminal GHHHHHHHH tag or the EcoRI and Stu sites of pBPH.IgG togenerate a C-terminal fusion with the Fc region of human IgG1. VectorspBPH.His.c and pBPH.IgG are derivatives of the baculovirus expressionvector pVL1393 (Pharmingen). A control Fc or His-tagged protein wasconstructed in a similar way be C-terminally linkingpancreatitis-associated protein (175 amino acid) to the Fc portion ofthe human IgG1 or a his-8 tag.

The fusion proteins were expressed in High 5 cells using themanufacturer's recommended procedure (Invitrogen). In brief, the DNAconstructs were co-transfected with BaculoGold Baculovirus DNA(Pharmingen) in a 7:1 ratio into adherent Sf9 cells. Cells wereincubated at 28° C. for 4 days and the supernatant was harvested. Thetransfection supernatant was amplified and was subject to affinitypurification by either protein A-Sepharose beads (Pharmacia) for Fcfusion proteins or Ni-NTA agarose beads (QIAGEN) for His-taggedproteins.

To examine the protein expression, SDS-PAGE analysis was performed onthe affinity purified recombinant proteins under non-reducing andreducing conditions, followed by silver staining.

Example 28 Induction of IL-6 and TNF-α Release by IL-17B (PRO1031),IL-17C (PRO1122)and IL-17E (PRO10272

Using the procedure outlined in Yao et al., J. Immunol. 155: 5483 (1995)for IL-6 release, human foreskin fibroblast cells (ATCC CRL-2091) werecultured in MEM media (10% FBS) with the test cytokine. After incubationfor 18 hours at 37° C. and 5% CO₂, conditioned media were assayed forIL-6 using an ELISA kit (R&D Systems). For TNF-α secretion, humanleukemia monocytic THP-1 cells were cultured in RPMI media (10% FBS)with test cytokine. After incubation for 18 hour at 37° C. and 5% CO₂,conditioned media were quantitated for TNF-α using and ELISA assay kit(R&D Systems).

Human foreskin fibroblast cells (ATCC) were separately cultured in MEMmedia (10% FBS) in the presence of IL-17B (PRO1031), and IL-17C(PRO1122). After incubation for 18 hours at 37° C. and 5% CO₂,conditioned media were assayed for IL-6 using an ELISA kit (R&DSystems). In contrast to the high level of IL-6 induced by IL-17, bothIL-17B (PRO1031) and IL17C (PRO1122) failed to stimulate IL-6 secretionin fibroblast cells (as shown in FIG. 36A).

Using the procedure outlined in Yao et al., Cytokine, 9: 794 (1997), ahuman leukemic monocytic cell line, THP-1, was used to assay for thestimulation of TNF-α release by IL-17, IL-17B (PRO1031) and IL-17C(PRO1031) by culturing in RPMI media (10% FBS). After incubation for 18hour at 37° C. and 5% CO₂, conditioned media were quantitated for TNF-αusing an ELISA assay kit (R&D Systems). While IL-17 induced only a lowlevel of TNF-α in THP-1 cells, both IL-17B and IL-17C (as Fc fusionproteins) stimulated TNF-α production in THP-1 cells (as shown in FIG.36B). A control Fc fusion protein had no effect.

In order to further characterize the stimulation of TNF-α release byIL-17B and IL-17C, the time course and concentration dependence of theresponse were assayed in THP-1 cells. FIG. 37 illustrates that IL-17Band IL-17C stimulate the release of TNF-α in a time- andconcentration-dependent manner. The EC50 for IL-17B stimulation is 2.4nM, while the EC50 for IL-17C is 25 nM.

While the IL-17B and IL-17C preparations used in these experimentscontained undetectable level of endotoxin (less than 1 EU/ml),additional control experiments were performed to confirm that the TNF-αrelease from THP-1 cells was real and not artifactual. The IL-17B andIL-17C activities were unaffected by polymyxin B treatment and wereabolished by heat treatment, further supporting the notion that theproteins themselves were responsible for the activities and not anycontaminating endotoxin.

Preparations of IL-17E were used to study the effect on IL-6 productionin human articular cartilage. Human articular cartilage was removed fromhuman patients during surgery for joint replacement and cartilageexplants were prepared as described in EXAMPLE 30 below. As shown inFIG. 46C., IL-17E was observed to induce IL-6 production in humanarticular cartilage. Human articular cartilage from diseased joints wascultured in media alone (−) or in combination with IL-17E at 0.1 nM, 1nM or 10 nM concentrations and matrix synthesis was determined by anELISA kit (R&D Systems) as described above. In contrast to the bothIL-17B (PRO1031) and IL17C (PRO1122) which failed to stimulate IL-6secretion in fibroblast cells (shown in FIG. 36A), IL-17E induced theproduction of IL-6, thereby exhibiting biological effects similar toIL-17 with respect to IL-6 release. IL-17 at 10 nM induced theproduction of IL-6 in human articular cartilage at comparable elevatedlevels (not shown).

Example 29 Fluorescence-Activated Cell Sorter (FACS) Analysis of Bindingto THP-1 Cells by IL-17, IL-17B and IL-17C Fusion Proteins

THP-1 cells (5×10⁵) were pre-incubated in PBS containing 5% horse serumat 4° C. for 30 minutes to block non-specific binding. IL-17, IL-17B.Fc,IL-17C.Fc, or control Fc (1 mg each) were added and incubated with theTHP-1 cells in a volume of 0.25 ml on ice for 1 hour. For the IL-17binding experiment, primary anti hIL-17 antibody (1:100 dilution) andsecondary goat anti-mouse antibody conjugated to FITC (JacksonImmunology Lab, 1:100 dilution) were added sequentially with 30-60minutes incubation and extensive washes before each addition. For the Fcfusion proteins, the cells were stained with FITC conjugated goatanti-human IgG (Fc specific, Jackson Immunology Lab, 1:100 dilution).After thorough washes, a minimum of 5,000 cells were analyzed using aFACScan (Becton Dickinson).

The resulting of the above procedure was that both IL-17B and IL-17C Fcfusion proteins displayed binding to THP-1 cells compared with a controlFc fusion protein (as shown in FIG. 38).

Example 30 Articular Cartilage Explant Assay For IL-17, IL-17C, andIL-17E A. Introduction:

As mentioned previously, IL-17 is likely to play a role in theinitiation or maintenance of the proinflammatory response. IL-17 is acytokine expressed by CD4⁺ T cells and induces the secretion ofproinflammatory and hematopoietic cytokines (e.g., IL-1b, TNF-α, IL-6,IL-8, GM-CSF) in a number of cell types including synoviocytes andmacrophages [Aarvak et al., J. Immunol., 162:1246-1251 (1999); Fossiezet al., J. Exp. Med., 183: 2593-2603 (1996); Jovanovic et al., J.Immunol., 160:3513-3521 (1998)]. In the presence of IL-17, fibroblastssustain the proliferation of CD34⁺ hematopoietic progenitors and inducetheir preferential maturation into neutrophils. As a result, Il-17 mayconstitute an early initiator of the T cell-dependent inflammatoryreaction and be part of the cytokine network which bridges the immunesystem to hematopoiesis.

Expression of IL-17 has been found in the synovium of patients withrheumatoid arthritis, psoriatic arthritis, or osteoarthritis, but not innormal joint tissues. IL-17 can synergize with the monocyte-derived,proinflammatory cytokines IL-1b or TNF-α to induce IL-6 and GM-CSF. Byacting directly on synoviocytes, IL-17 could enhance secretion ofproinflammatory cytokines in vivo and thus exacerbate joint inflammationand destruction.

While the role of IL-17 in inflammation has been studied by a number ofinvestigators, the direct effects of IL-17 on articular cartilage havenot been well-characterized. To further understand the possible role ofIL-17, Applicants have tested the effects of IL-17 on cartilage matrixmetabolism. In light of the known catabolic effects of nitric oxide (NO)on cartilage, and the existence of high levels of NO in arthriticjoints, NO production was also measured. In addition, studies with twoother novel IL-17 homologs IL-17C (PRO1122) and IL-17E (PRO10272) wereundertaken to determine if similar physiological effects could be seen.

B. Methods:

Articular Cartilage Explants Used in Studies with IL-17, IL-17C andIL-17E:

In studies using test proteins IL-17 and/or IL-17C, themetacarpophalangeal joint of a 4-6 month old female pigs was asepticallydissected, and articular cartilage is removed by free-hand slicing in acareful manner so as to avoid the underlying bone. Similarly in studieswith both IL-17 and IL-17E, human articular cartilage explants wereremoved from human patients during surgery for joint replacement. Thecartilage from either source was treated similarly for the assays asdescribed below. The cartilage was minced and cultured in bulk for atleast 24 hours in a humidified atmosphere of 95% air 5% CO₂ in serumfree (SF) media (DME/F12 1:1) with 0.1% BSA and antibiotics. Afterwashing three times, approximately 80 mg of articular cartilage wasaliquoted into micronics tubes and incubated for at least 24 hours inthe above SF media. IL-17 and IL-17C test proteins were then added at 1%either alone or in combination with IL-1a (10 ng/ml) in studies withporcine cartilage. For IL-17 and IL-17E studies, human articularcartilage was treated with various concentrations of IL-17 and IL-17E(0.1 nM, 1 nM and 10 nM, respectively). Control (−) tubes containedmedia only. Media was harvested and changed at various timepoints (0,24, 48, 72 hours) and assayed for proteoglycan content using the1,9-dimethyl-methylene blue (DMB) colorimetric assay described inFarndale and Buttle, Biochem. Biophys. Acta, 883:173-177 (1985). Afterlabeling (overnight) with ³⁵S-sulfur, the tubes were weighed todetermine the amount of tissue. Following an overnight digestion, theamount of proteoglycan remaining in the tissue as well as proteoglycansynthesis (³⁵S-incorporation) is determined.

Measurement of NO production: The assay is based on the principle that2,3-diaminonapthalene (DAN) reacts with nitrite under acidic conditionsto form 1-(H)-naphthotriazole, a flourescent product. As NO is quicklymetabolized into nitrite (NO2-1) and nitrate (NO3-1), detection ofnitrite, is one means of detecting (albeit undercounting) the actual NOproduced. 10 mL of DAN (0.05 mg/mL in 0.62 M HCl) is added to 100 mL ofsample (cell culture supernatant), mixed, and incubated at roomtemperature for 10-20 minutes. Reaction is terminated with 5 mL of 2.8NNaOH. Formation of 2,3-diaminonaphthotriazole was measured using aCytoflor flourescent plate reader with excitation at 360 nm and emissionread at 450 nm. For optimal measurement of flourescent intensity, blackplates with clear bottoms were used.

C. Results and Discussion:

IL-17 was observed to both increase the release of and decrease thesynthesis of proteoglycans in both human and female pig articularcartilage explants (porcine extracts results are shown in FIG. 39).Moreover, this effect was additive to the effect observed from IL-1a.The effects of IL-17 are not mediated by the production of nitric oxide,nor does inhibition of nitric oxide release augment matrix breakdown(see FIGS. 40 to 42). IL-17C (PRO1122) was also observed to increasematrix breakdown and inhibit matrix synthesis in female pig articularcartilage explants (FIG. 43). Thus, expression of PRO1122 is likely tobe associated with degenerative cartilagenous disorders.

In addition, the effect of IL-17 and IL-17E (PRO10272) on humanarticular cartilage explants was studied wherein both IL-17 and IL-17Ewere observed to inhibit matrix synthesis in human cartilage at variousconcentrations. Human articular cartilage from diseased joints wascultured in media alone (-) or in combination with either IL-17 orIL-17E at 0.1 nM, 1 nM or lOnM concentrations, respectively, and matrixsynthesis determined by measuring ³⁵S-sulfate uptake as described above(see FIG. 46A. for results with IL-17E) Likewise, the effects of IL-17and IL-17E on nitric oxide production were studied. Human articularcartilage was cultured in media alone (−) or in combination with eitherIL-17 or IL-17E at 0.1 nM, 1 nM or 10 nM concentrations and theproduction of nitric oxide measured as described in Part B. Methods.(Measurement of NO production). FIG. 46B. demonstrates the effect ofIL-17E on nitric acid production wherein various concentration of IL-17Einduces nitric oxide production in comparison to the controls (−). IL-17gave comparable increased levels over the controls (not shown). Thus,expression of PRO10272 is also likely to be associated with degenerativecartilagenous disorders.

In conclusion, IL-17, IL-17C and IL-17E likely contribute to loss ofarticular cartilage in arthritic joints, and thus inhibition of itsactivity might limit inflammation and cartilage destruction. IL-1a andIL-17 have similar yet distinct activities, due to their use ofdifferent receptors and overlapping downstream signaling mechanisms.

Given the findings of the potent catabolic effects of IL-17 on articularcartilage explants and the homology of IL-17E (PRO10272) and IL-17C(PRO1122) to IL-17, antagonists to any or all of these proteins may beuseful for the treatment of inflammatory conditions and cartilagedefects such as arthritis.

Finally, it is well known that growth factors can have biphasic effectsand that diseased tissue can respond differently than normal tissue to agiven factor in vivo. For these reasons, antagonists or agonists (e.g.,the proteins themselves) of IL-17E (PRO10272), IL-17C (PRO1122), orIL-17, may be useful for the treatment of inflammatory conditions andjoint disorders such as arthritis.

Example 31 Inflammatory Bowel Disease (IBD): Expression of IL-17 Familyin Mouse Model of IBD

Mice deficient in the cytokine receptor CRF2-4/IL-10Rb developspontaneous and progressive colitis that resembles the human conditionof inflammatory bowel disease (IBD). This phenotype has been previouslyreported (Spencer et al., J. Exp. Med., 187:571-578 [1998]). To examinethe role of expression of IL-17 family members in this model of IBD,colons were harvested from normal (wild type “WT”) mice and from CRF2-4deficient mice. Colons from CRF2-4 deficient mice were sub-categorizedinto specimens exhibiting mild IBD and specimens exhibiting moreadvanced severe IBD. RNA was isolated from the colon samples and therelative expression of IL-17 family members was determined byquantitative PCR (Taqman™). FIG. 44 demonstrates the relative expressionof IL-17, IL-17E (DNA147531-2821), IL-17B (DNA59294-1381-1), and IL-17D(DNA173894-2947) represented by -delta CT relative to GAPDH. Theexpression of IL-17E markedly decreases in more advanced severe IBDcompared to expression levels in normal (wild type “WT”) mice. Incontrast, increased expression values of IL-17 were observed in mild tosevere IBD. Thus, IL-17E may serve as a marker for this inflammatorycondition.

Example 32 IL-17D Expression in Mouse Model of Stroke

IL-17D (DNA173894-2947) expression was examined in a murine experimentalmodel of stroke. The right common carotid artery (RCCA) of C57B 1/6 malemice was isolated via a midline incision. A loose tie was placed aroundthe vessel. The middle cerebral artery (MCA) was visualized by forming acranial window in the skull at the level of the rhinal fissure. At theappointed time, the MCA and the RCCA were occluded for a 45 minuteischemic period with 11-0 suture and 6-0 suture, respectively. Followingthis ischemic insult, the RCCA and MCA sutures were untied to allowreperfusion of the MCA territory. The relative expression of IL-17D wasdetermined by quantitative PCR (Taqman™) using RNA isolated fromischemic cortex at five time points following reperfusion (3, 6, 12, 24and 72 hours) and compared with the expression of IL-17D observed in RNAisolated from control non-ischemic tissue. FIG. 45 depicts the resultsof this study. As shown, IL-17D expression decreases rapidly followingstroke when examined over the five illustrated time points.

Example 33 IL-17F (PRO20110) Stimulation of Cytokine Production andCartilage Matrix Turnover A. Methods (1) Protein Expression andPurification

An IL-17F cDNA clone was isolated from a human cDNA library andsequenced in its entirety. Essentially the same strategy was used toexpress and purify IL-17F (PRO20110), IL-17, IL-17E (PRO10272), IL-17Rextracellular domain (ECD) (residues 1-288), and IL-17RH1 (PRO5801) ECD(residues 1-275). DNA containing the coding region for each protein ofinterest was first amplified by PCR, then subcloned into pET15b(Novagen) via NdeI and BamIII/BglII sites in order to introduce anN-terminal His-tag and thrombin cleavage site. After another PCR step,the coding region was subcloned into the baculovirus transfer vectorpAcGP67B (PharMingen) via BamHI/BglII and NotI sites. The transfervector was then co-transfected with BaculoGold DNA (PharMingen) into Sf9cells, and recombinant virus was isolated and amplified in Sf9 cell to2×10⁸ pfu/ml.

For protein production, Hi5 cells were infected with amplifiedbaculovirus. After three days in culture at 27° C., the medium washarvested by centrifugation. 50 mM Tris, pH 7.5, 1 mM NiCl₂, 5 mM CaCl₂,1 μM PMSF and 0.01% NaN₃ were added and the pH was adjusted to 7.0. Themedium was filtered and loaded onto a Ni-NTA (Qiagen) column. The columnwas washed with 50 mM sodium phosphate, pH 7.0, 500 mM NaCl, 10 mMimidazole, then eluted with 250 mM imidazole in the same buffer.Fractions containing the protein of interest were pooled and dialyzedinto PBS, pH 6.5, together with 1 unit/mg thrombin (Calbiochem)overnight at 4° C. The protein sample was then concentrated and thethrombin and His-tag were removed by purification over a Superdex-75column in 50 mM sodium phosphate, pH 6.0, 500 mM NaCl. IL-17, IL-17E andIL-17F all migrated as dimers, whereas IL-17R and IL-17RH1 migrated asmonomers on this column. IL-17, IL-17E and IL-17F exist predominantly asdisulfide-bonded dimers as evidenced by comparison of reducing andnon-reducing SDS PAGE. Relevant fractions were pooled and dialyzed into50 mM sodium phosphate, pH 6.0, 150 mM NaCl. For crystallization ofIL-17F, the protein was instead dialyzed into 25 mM Bis-Tris Propane, pH6.0, 100 mM NaCl and concentrated to 10 mg/ml. N-terminal sequencingconfirmed the identities of all purified proteins and indicated thatthey each have the expected additional four amino acids (GSHM) at theN-terminus introduced by the vector's cloning site. Mass spectrometryshowed evidence of glycosylation of all proteins, with IL-17F containingapproximately 3 kDa carbohydrate per dimer.

(2) Expression in Activated T Cells

CD4⁺ and CD8⁺ T cells were isolated from human peripheral bloodmononuclear cells using MACS (Miltenyi Biotec, Inc. Germany) accordingto manufacturer's instruction and confirmed by FACS analysis. Thepurified T cells were treated with PMA (10 ng/ml) plus ionomycin (500ng/ml) for 4 hours. Total RNA samples were prepared using TRIZOL Reagentaccording to manufacturer's instructions (GIBCO-BRL). IL-17F transcriptlevel was measured by real-time quantitative RT-PCR.

(3) Articular Cartilage Explants

IL-17 and IL-1α were purchased from R&D systems and resuspended inbuffer (PBS with 0.1% BSA) prior to use. The metacarpo-phalangeal jointof 4-6 month old female pigs was aseptically opened, and articularcartilage was dissected free of the underlying bone. The cartilage waspooled, minced, washed and cultured in bulk for at least 24 hours in ahumidified atmosphere of 95% air and 5% CO₂ in serum-free low glucose50:50 DMEM:F12 media with 0.1% BSA, 100 U/ml penicillin/streptomycin(Gibco), 2 mM L-glutamine, 1×GHT, 0.1 mM MEM Sodium Pyruvate (Gibco), 20μg/ml Gentamicin (Gibco), 1.25 mg/L Amphotericin B, 10 μg/ml transferrinand 5 μg/ml Vitamin E. Approximately 50 mg of articular cartilage wasaliquoted into Micronics tubes and incubated for at least 24 hours inabove media before changing to media without transferrin and Vitamin E.Test proteins were then added. Media was harvested and changed atvarious time points (0, 24, 48, 72 hr). Human knee articular cartilage,received from the National Resource Center (Philadelphia, Pa.), wascultured and treated in explants as above for porcine cartilage.

To measure proteoglycan breakdown, media harvested at various timepoints were assayed for amount of proteoglycans using the1,9-dimethlymethylene blue (DMB) colorimetric assay (Farndale et al.,Biochem. Biophys. Acta 883:173-177 (1992)). Chondroitin sulfate (Sigma)ranging from 0.0 to 5.0 μg was used to make the standard curve. Tomeasure effects on proteoglycan synthesis, ³⁵S-sulfate (to a finalconcentration of 10 μCi/ml) (ICN Radiochemicals, Irvine, Calif.) wasadded to the cartilage explants at 48 hr. After an overnight incubationat 37° C., media was saved for measurements of nitric oxide orproteoglycan content. Cartilage pieces were washed two times usingexplant media. Digestion buffer containing 10 mM EDTA (pH 8.0), 0.1 Msodium phosphate (pH 6.5) and 1 mg/ml proteinase K (Gibco BRL) was addedto each tube and incubated overnight in a 50° C. water bath. The digestsupernatant was mixed with an equal amount of 10% w/v cetylpyridiniumchloride (Sigma). Samples were spun at 1000 g for 15 min. Thesupernatant was removed, and 500 μL formic acid (Sigma) was added to thesamples to dissolve the precipitate. Solubilized pellets weretransferred to scintillation vials containing 10 ml scintillation fluid(ICN), and samples were read in a scintillation counter. RecombinantIL-17 (prepared identically to IL-17F) had the same activity as IL-17obtained commercially, indicating that the addition of amino acids atthe N-terminus does not disrupt IL-17 function, and suggesting that theactivity measured for recombinant IL-17F represents native IL-17Factivity.

(4) Cytokine ELISAs

Conditioned media from explant cultures at 48 hr were diluted 15-fold(porcine IL-6) or 150-fold (human IL-6) and used for assays. G-CSF andIL-8 production from cultured fibroblasts was determined as recommendedby assay manufacturer (R&D Systems).

B. Results and Discussion (1) Identification of IL-17F (PRO20110)

IL-17 is now recognized as the prototype member of an emerging family ofcytokines (Yao et al., Immunity 3:811-821 (1995); Yao et al., Immunol.155:5483-5486 (1995); Li et al. Proc. Natl. Acad. Sci. USA 97:773-778(2000); Shi et al., J. Biol. Chem. 275:19167-19176 (2000); and Lee etal. J. Biol. Chem. 276:1660-1664 (2001). The gene encoding human IL-17Fis located adjacent to IL-17 (human genomic sequence in cloneRP11-935B23; Genbank accession number AL355513). A cDNA corresponding toIL-17F was isolated (SEQ ID NO:9) and found to encode a protein of 163amino acids (including a 30 residue signal sequence) (PRO20110) (SEQ IDNO:10) bearing 44% amino acid sequence identity to IL-17 (SEQ ID NO:40)and identity with a clone isolated by Jacobs et al., WO97/07198-A2(1997). Other members of the IL-17 family share a more limited 15-27%amino acid sequence identity suggesting that IL-17 and IL-17F form adistinct subgroup within the IL-17 family.

(2) IL-17F mRNA Expression

The expression of IL-17F in various human tissues was examined byNorthern blot analysis (see FIG. 24). IL-17F mRNA levels were alsomeasured in purified T cells. Little message was present in unstimulatedCD4⁺ or CD8⁺ T cells, however, IL-17F mRNA was dramatically induced inactivated CD4⁺ T cells (FIG. 47A). Interestingly, a significant level ofexpression was also detected in activated CD8⁺ T cells. Thus, IL-17Fexpression is rare, but is highly induced in activated CD4⁺ T cells.

(3) IL-17F Stimulates Cytokine Production

The sequence similarity between IL-17F (PRO20110; SEQ ID NO:10) andIL-17 (SEQ ID NO:40) as well as their expression within activated Tcells raises the possibility that they may possess similar biologicalactivities. Studies were performed to investigate whether IL-17F couldinduce production of cytokines known to be regulated by IL-17 (Fossiezet al., J. Exp. Med. 183:2593-2603 (1996)). Primary human fibroblaststreated with IL-17F exhibited greatly elevated production of G-CSF andIL-8 (as shown in FIG. 47B and FIG. 47C). Similar responses wereobserved with various cells lines tested (not shown) suggesting thatIL-17F possesses broad ability to induce the production of moleculesknown to be regulated by IL-17.

(4) Effect of IL-17F (PRO20110) on Cartilage Matrix Turnover

As a potent proinflammatory cytokine produced by activated T cells,IL-17 has been suggested to play a role in inflammatory disorders suchas rheumatoid arthritis. To determine whether IL-17F might be capable ofmediating similar affects on cartilage matrix metabolism, porcine andhuman articular cartilage explants were treated with a range of IL-17Fconcentrations, and proteoglycan release and synthesis were measured. Inboth systems IL-17F induced significant cartilage matrix release (asshown in FIG. 48A and FIG. 48D) and inhibited new cartilage matrixsynthesis (as shown in FIG. 48B and FIG. 48E) in a dose-dependentmanner. These effects were on the same order of magnitude as that of theknown catabolic cytokine, IL-1α. At higher concentrations (10 nM),IL-17F and IL-17 showed equal potency on human articular cartilagematrix turnover (as shown in FIG. 48D and FIG. 48E). Thus, IL-17F candirectly regulate cartilage matrix turnover; however, the potency ofIL-17F relative to that of IL-17 depends on the species tested and mayrelate to receptor affinity.

Previous results showed that IL-17 substantially induced expression ofIL-6, a potent regulator of bone turnover, and IL-8 in human cartilage,but did not change the levels of IL-2, IL-4, IL-5, IFN-γ or TNF-α. Inboth human and porcine articular cartilage, IL-17F could also induceIL-6 production in a dose-dependent manner (as shown in FIG. 48C andFIG. 48F). In particular, IL-17F, like IL-17, induced IL-6 atconcentrations (0.1 nM and 1 nM) at which no significant change inmatrix turnover or synthesis was observed (see FIG. 48C and FIG. 48F).In addition, IL-17F was less potent than IL-17 in both porcine and humancartilage in terms of IL-6 production in contrast to the similarity inpotency on human cartilage matrix turnover.

The IL-17s constitute an emerging family of cytokines. The potentbiological actions that have been observed to date suggest the potentialfor members of this family to contribute to immune disorders. Initialreports have pointed to a clear association of IL-17 with rheumatoidarthritis (Chabaud et al. Arthritis Rheum. 42:963-970 (1999); Kotake etal., J. Clin. Invest., 103:1345-1352 (1999); Ziolkowska et al., J.Immunol. 164:2832-2838 (2000)), a disease characterized by infiltrationof leukocytes, synovitis, pannus formation, and skeletal destruction(Arend, W. P., and Dayer, J. M., Arthritis Rheum. 33:305-315 (1990)). Inhumans, activated T cells likely play a key role in the disease processthrough both direct and indirect mechanisms (Kingsley, G. H., andPanayi, G. S., Clin. Exp. Rheumatol. 15:S3-S14 (1997); Miossec, P.,Curr. Opin. Rheumatol. 12:181-185 (2001)). More specifically, activatedT cells stimulate other cells such as macrophages and fibroblasts torelease cytokines which can then amplify the local immune response andpromote synovitis. The present inventors show that IL-17F is alsoproduced by activated T cells and can have direct effects on articularcartilage matrix turnover and IL-6 production in the absence ofinflammatory cells, thus suggesting that IL-17F may also be able topromote skeletal tissue distruction.

In conclusion, IL-17 and IL-17F (PRO20110) likely contribute to loss ofarticular cartilage in arthritic joints, and thus inhibition of itsactivity might limit inflammation and cartilage destruction. IL-1a andIL-17 have similar yet distinct activities, due to their use ofdifferent receptors and overlapping downstream signaling mechanisms.

Given the findings of the potent catabolic effects of IL-17F (PRO20110)on articular cartilage explants and the homology of IL-17F (PRO20110) toIL-17, antagonists to these proteins may be useful for the treatment ofinflammatory conditions and cartilage defects such as arthritis.

Example 34 IL-17F Receptor Binding; IL-17F Structure Determination A.Methods (1) Binding Measurements

The kinetics and affinity of IL-17, IL-17E (PRO10272), or IL-17F(PRO20110) binding to IL-17R or IL-17RH1 (PRO5801) were determined bySPR measurements on a Pharmacia BlAcore 1000 instrument (PhamaciaBiosensor, Piscataway N.J.). IL-17 ligand or receptor was immobilizedonto a flow cell of a CM5 sensor chip via random coupling to aminogroups, N-hydroxysuccinimide chemistry, by using a protocol developed bythe manufacturer. An immobilization level of about 500 resonance units(RU) was obtained for IL-17R, IL-17RH1, and IL-17F, whereas IL-17 andIL-17E immobilization levels were 1200 and 1500 RU, respectively. Astrong signal was obtained for IL-17R binding to immobilized IL-17.However, when IL-17R was immobilized, only a weak signal was obtainedfor IL-17 binding suggesting that receptor immobilization inactivatesthe binding site. After blocking unreacted sites with ethanolamine,binding measurements were performed using a flow rate of 25 μL/min.Sensorgrams were obtained for a series of six, two-fold serially dilutedprotein solutions. The highest concentration used was 1000 or 500 nMprotein and the solutions were prepared in the running buffer, PBScontaining 0.05% Tween-20. The sensor chip surface was regeneratedbetween binding cycles by injection of a 25 μL aliquot of 0.1 M aceticacid, 0.2 M NaCl, pH 3 to elute non-covalently bound protein.Sensorgrams were evaluated according to a 1:1 binding model bynon-linear regression analysis using software supplied by themanufacturer. In separate experiments to measure competition betweenIL-17 variants for binding receptors, a fixed concentration of receptorwas incubated with a varied concentration of IL-17 protein followed byinjection of this mixture onto a flow cell having immobilized IL-17protein. The amount of bound receptor was determined from the resonancesignal obtained after completion of the association phase.

(2) Crystallography

IL-17F crystallized as hexagonal plates in hanging drops over a wellsolution containing 1.0 M lithium sulfate, 0.5 M ammonium sulfate, 1%ethanol, and 100 mM sodium citrate, pH 5.6, at 19° C. Crystals wereharvested into an artificial mother liquor consisting of the wellsolution without ethanol. Prior to data collection, crystals wereimmersed in artificial mother liquor with 20% glycerol and flash-cooledin liquid nitrogen. Initial data were collected on an in-house rotatinganode generator with CuK∀ radiation and the space group was found to beP6₁ or P6₅, with two dimers in the asymmetric unit. For phasing,crystals were derivatized by soaking for 6 hr in artificial motherliquor supplemented with 2 mM thimersol. A native data set and a threewavelength Hg MAD (Multiwavelength Anomalous Diffraction) experimentwere collected at beam line 9-2 at the Stanford Synchrotron RadiationLaboratory. The data sets were processed using the programs in the HKLpackage (Otwinowski, Z., and Minor, W., Methods Enzymol. 176:307-326(1997)). Structure determination was carried out using the CCP4 suite ofprograms (CCP4, Acta Cryst. D50:760-763 (1994)). Patterson mapsindicated the presence of several well-ordered Hg atoms whose locationwere determined using the program Rantan. Phase refinement was carriedout with MLPHARE. Examination of DM-modified maps indicated that thespace group was P6₅ and revealed the non-crystallographic symmetry (NCS)operators. Each protomer bound a single thimerosal at an equivalent,NCS-related site.

The initial structure was built into a four-fold NCS-averaged andsolvent flattened experimental map and was refined using the programsREFMAC_(—)4.0 (CCP4, Acta Cryst. D50:760-763 (1994)) and Brünger, A. T.(1992), X-PLOR Manual, Version 3.1 (New Haven, Connecticut: YaleUniversity) as modified by Molecular Simulations, Inc. Reflectionssequestered for calculating the free R-value were chosen in thinresolution shells. A maximum likelihood target function, an overallanisotropic correction, and a real-space bulk-solvent correction wereused during positional refinement, simulated annealing, and isotropictemperature factor refinement. Initial refinement was done against the2.65 Å remote data set but disorder around the Hg sites proved difficultto model so final refinement was carried out against the 2.85 Å nativedata set, using the same set of free R reflections. In the final model,the four vector-derived residues, residues 1 to 8, and residues 128-133are disordered in protomers A, B and X, while residues 1-6 and 130-133are disordered in protomer Y. In the XY dimer, an internal loop(residues X20-X23 and Y20-Y25) is disordered; this same loop is poorlyordered in the AB monomers. A Ramachandran plot shows that 90% of allnon-glycine, non-proline residues are in the most favored regions, 9.2%in the additional allowed regions, 0.7% (3 residues) in the generouslyallowed regions, and no residues in the disallowed regions. Datacollection and refinement statistics in Table 8. The coordinates forIL-17F have been deposited in the Protein Data Bank and access code yetto be assigned. The programs areaimol and resarea (CCP4, Acta Cryst.D50:760-763 (1994)) were used for accessible surface area calculations.The programs Molscript (Kraulis, P. J., J. Appl. Cryst. 103:1345-1352(1999)); Raster3D (Merrit, E. A., and Murphy, M. E. P., Acta Cryst.D50:869-873 (1994)); Insight97 (MSI) and Grasp (Nichols et al., Proteins11:281-296 (1991)) were used for analysis and to make FIGS. 49, 51 and52.

B. Results and Discussion (1) Receptor Binding

Surface plasmon resonance (SPR) was used to determine whether IL-17F(PRO20110) binds the extracellular domains (ECDs) of either of the tworeceptors IL-17R (designated PRO1) and IL-17RH1 (PRO5801) reported tobind IL-17 proteins. However, no binding of either IL-17R or IL-17RH1(up to 1 and 0.5 μM, respectively) was observed to immobilized IL-17F.In contrast, IL-17R bound immobilized IL-17 with a modest bindingaffinity (see Table 7 below), consistent with previous reports on theaffinity for this interaction (Yao et al., Cytokine 9:794-800 (1997)).Likewise, IL-17RH1 showed high affinity binding to IL-17E (Table 7),consistent with the potency observed for induction of IL-8 release fromcells (Lee et al., J. Biol. Chem. 276:1660-1664 (2001)). Furthermore, nobinding was observed between IL-17RH1 and IL-17 and between IL-17E andIL-17R as expected (Shi et al., J. Biol. Chem. 275:19167-19176 (2000);Lee et al., J. Biol. Chem. 276:1660-1664 (2001)).

To test whether the lack of IL-17R or IL-17RH1 binding to IL-17F couldbe the result of immobilization-linked activation, IL-17F/receptorbinding was tested in competition experiments. In these experiments afixed concentration of IL-17R (500 nM) or IL-17RH1 (31 nM) was incubatedwith a varied concentration of ligand, and then injected over the IL-17or IL-17E surface. While soluble IL-17 could efficiently block bindingof IL-17R to immobilized IL-17, no competition was observed with 2 μMIL-17F. Furthermore, 1.3 μM IL-17F could not block binding of IL-17RH1to immobilized IL-17E, although binding was completely inhibited bysoluble IL-17E. These results indicated that IL-17F does not bind withhigh affinity to the purified, monomeric, ECD of either IL-17R orIL-17RH1. As shown in EXAMPLE 20 (FIG. 35), IL-17F ligand has been shownto bind to novel IL-17RH2 receptor (PRO20040).

Although IL-17F appears to have activity related to that of IL-17,IL-17F does not bind IL-17R with high affinity in vitro. However,enhanced binding of IL-17F to Cos cells transfected with IL-17R can bedetected (not shown), suggesting that IL-17F may be able to utilizeIL-17R, but only in combination with additional, yet unidentifiedcomponents to form a high affinity signaling complex. A similarmechanism has been postulated for IL-17 to explain the discrepancybetween receptor affinity and the potency of its biological activity(Yao et al., Cytokine 9:794-800 (1997)). The results presented hereinsuggest that regardless of the receptor(s) involved, IL-17F signalingresults in similar downstream activities as stimulation of IL-17R byIL-17.

TABLE 7 Kinetics and Affinity of Receptor Binding to Immobilized IL-17and IL-17E K_(on) × 10⁻⁵ K_(off) × 10⁴ K_(p) Immobilized Protein Ligand(M⁻¹ s⁻¹) (s⁻¹) (nM) IL-17 IL-17R 0.093 6.7 72 IL-17E IL-17RH1 6.7 7.01.1 IL-17RH1 IL-17E 4.3 6.2 1.4

(2) Structure Determination of IL-17F

The structure of human IL-17F was solved by multiwavelength Hg anomalousdiffraction methods and was refined to an R_(free) and R_(cryst) of28.8% and 23.3%, respectively, at 2.85 Å resoultion (see Table 8 below).The core of an IL-17F protomer is composed of two pairs of antiparallelβ-strands; one pair includes strands 1 (residues 52-59) and 2 (residues66-73 and 77-79), while the other includes strands 3 (89-103) and 4(110-125). Strand 2 is interrupted by a short stretch of irregularβ-structure. Two disulfide bridges (Cys 72/Cys 122 and Cys 77/Cys 124)connect strands 2 and 4. A third disulfide (Cys 17/Cys 107) connects theloop between strands 3 and 4 of one protomer to the N-terminal extensionof the adjacent protomer forming extensive dimer contacts (as discussedbelow). This N-terminal extension also contains a β-strand (strand 0,residues 25-32), which hydrogen-bonds to strand 3′ on the otherprotomer, and a small α-helix (residues 43-48). Additional electrondensity was observed at Asn 53, consistent with glycosylation of thisresidue as was expected from sequence analysis and characterization ofthe purified protein.

This structure reveals that IL-17F is a distant homolog of the cystineknot family of proteins (McDonald, N. Q., and Hendrickson, W. A., Cell73:421-424 (1993)), named for its unusual cystine connectivity (FIG.49). The cystine knot is characterized by two sets of paired β-strands(strands 1 and 2 and strands 3 and 4) that are connected by disulfidelinkages between strands 2 and 4 (FIG. 49A, inset). A third disulfidebridge passes through this macro-cycle to connect strands 1 and 3. Incontrast, IL-17F contains only two of the three distinctive cystinelinkages that give the family its name. In IL-17F, the Cys 72/Cys 122and Cys 77/Cys 124 disulfides form the macro-cycle of the typicalcystine knot. The third disulfide which would form the “knot” by passingthrough this macro-cycle is not present; instead, residues 50 and 89,which are located in the same three-dimensional space as the thirddisulfide in cystine-knot proteins, are serines in IL-17F. While Ser 50is in the same conformation as the corresponding cysteine in aknot-protein, Ser 89 is not. It is noteworthy that serines are conservedin these positions in all IL-17 family members (see FIG. 50), despitethe fact that the structure suggests the third disulfide could beaccommodated.

TABLE 8 Crystallographic Statistics Data Collection and MAD Phasing HgHg Hg Native Peak Inflection Remote Space Group P6₅ Unit Cell Constants(Å) a = 126.4, a = 126.8, b = 89.9   b = 90.0   Wavelength (Å) 0.9791.0067 1.0087 1.127 Resolution (Å) 2.85 2.8 2.8 2.65 I/Isig 7.7 11.411.2 9.1 Completeness (%) 100 (100) 98.9 (91.3) 98.8 (90.2) 99.9 (99.9)Rsym^(b)  8.8 (54.1)  5.8 (34.4)  5.9 (35.9)  6.4 (43.1) Reflectionsmeasured^(c) 141778 228788 228553 266735 Reflections unique^(c) 1929440450 40459 46851 Phasing power centric^(d) — 1.4 1.6 1.3 Phasing poweracentric^(d) — 4.3 3.2 4.3 Rcullis acentric^(d) — 0.76 0.7 0.8Refinement Resoloution (Å) 30-2.85 # Reflections 19246 R^(e) 0.233R_(free) 0.288 # protein atoms 3716 # carbohydrate atoms 84 3 waters 21Rmsd bonds (Å) 0.12 Rmsd bonded Bs 4.5 Rmsd angles (°) 1.7 ^(a)Numbersin parentheses refer to the highest resolution shell ^(b)R_(sym) = Σ|I −<I|/ΣI. <I> is the average intensity of symmetry related observations ofa unique reflection. ^(C)Bijvoet reflections are kept separate in the Hgstatistics ^(d)Phasing statistics are for reflections with F > 2σ ^(e)R= Σ|F_(o) − F_(c)|/ΣF_(o)

(1) Dimerization

IL-17F dimerizes in a parallel fashion similar to nerve growth factor(NGF) and other neutrophins (McDonald et al., Nature 354:411-414(1991)). However, the dimer interface is unusually large, burying atotal of 6800 Å² (or ˜3400 Å² per monomer) as compared to 3400 Å² total(˜1700 Å² per monomer) for NGF (PDB code 1WWW; Weismann et al. Nature401:184-188 (1999)). Approximately one third of the interface is formedby interactions between strands 3 and 4 of one monomer with the samestrands in the other monomer, analogous to the dimer interface seen inneutrophins. Unique to IL-17F, is the vast amount of surface area buriedby interactions involving the N-terminal extension (residues 8-48) ofeach protomer reaching across the canonical dimer interface and packingagainst various portions of the other protomer.

The overall backbone structure of the IL-17F dimer can be described as agarment where sheets 1/2 and 1′/2′ form the sleeves, the cystine knotdisulfides line the collar, and sheets 3/4 and 3′/4′ along with theN-terminal extensions form the body, which is finished off with the twothree-stranded sheets (involving strands 4/3/0′ and 0/3′/4′) forming askirt at the bottom (FIG. 49B; dimensions 65 Å×25 Å×30 Å). A strikingfeature on the surface of the molecule is an unusually large cavity (18Å×10 Å×10 Å deep) located at the dimer interface essentially positionedas pockets in the garment. The base of the cavity is formed by residuesin strands 3 and 3′ (Gln 95, Glu 96, Thr 97, and Leu 98 from bothchains) and 4 and 4′ (Lys 115′, Val 118, and Val 120′). Residues in theN-terminus line one side of the cavity (residues Arg 37, Val 38, Met40), while the other side is lined by residues from strand 1 (Tyr 54),strand 2 (Val 68, Glu 66), and the turn between these strands (Tyr 63and Pro 64). The peptide bond between Tyr 63 and Pro 64 is in theunusual cis conformation. Since this proline is conserved in all IL-17sequences and is always proceeded by a large hydrophobic residue (seeFIG. 50), it is unlikely that this peptide bond is in a cis conformationin all IL-17 family members. The mercury-containing compound,thimerosal, which was used to phase the structure, binds in the lowerend of the this cavity (as oriented in FIG. 51), occupying 30% of thespace.

The structural features discussed above demonstrate that an IL-17homolog (IL-17F) is a member of the cystine knot fold superfamily anddimerizes similarly to members of the NGF subfamily. IL-17 proteinsshare negligible sequence similarity with other members of thesuperfamily. For example, a structure-based sequence alignment of IL-17Fwith NGF reveals identity for only ten residues, including the fourcysteines conserved in the cystine knot motif (not shown). Limitedsequence conservation is typical of the cystine knot fold superfamily(McDonald, N. Q., and Hendrickson, W. A., Cell 73:421-424 (1993)).

The structure of IL-17F allows generalization to the other IL-17 familymembers. The cystine-knot fold including the location of the ∃-sheetsand the macro-cycle disulfide linkage should be preserved in all IL-17homologs (FIG. 50). In particular, IL-17 is so similar to IL-17F,sharing almost 50% sequence identity, that it is possible to predictwhere IL-17 and IL-17F will share surface features and where they willdiverge. FIG. 51 shows the molecular surface of IL-17F colored accordingto sequence identity with IL-17. The only extensive conserved patches onthe surface of IL-17F are on the flat face of each protomer (FIG. 51B)and on the area “above” the cavity (FIG. 51A). The conserved area on theprotomer face may represent either conserved features required formaintaining the structure or for the potentially recognizing commonbinding partners. The large cavity in the surface of IL-17F, thus isexpected to also be present in IL-17, but would be composed of bothconserved and variable residues.

In contrast, the sequences of IL-17B, IL-17C, and IL-17E divergesignificantly from IL-17F and IL-17, especially in the number andlocation of auxiliary cystine linkages and the length and sequence ofthe N-terminal extension. Despite this divergence, it is possible tomake several predictions about the disulfide connectivity and the effectit will have on the N-terminal extension in other family members. Forexample, IL-17B is secreted as a non-covalent dimer (both from CHO orinsect cells (Shi et al. J. Biol. Chem. 275:19167-19176 (2000) and datanot shown) indicating all eight cysteine residues are paired within asingle chain of the dimer. One of the two additional cysteines in IL-17B(Cys 103) is located between the two cysteines in strand 2 that areinvolved in the macro-cycle while the second additional cysteine is inthe turn between strands 3 and 4 (FIG. 50 and FIG. 49). Based on theassumption that the cystine knot fold is conserved in all IL-17homologs, the extra cysteine (Cys 103) in strand 2 of IL-17B would belocated too far away to bond to either of the cysteines in the strand3/4 loop. Therefore, Cys 103 of IL-17B must disulfide bond to Cys 64 inthe N-terminal extension, leaving the two cysteines in the strand 3/4loop to bond to each other. In order for these interactions to takeplace, the N-terminal extension must be in a radically differentconformation in IL-17B than in IL-17F. This is reasonable since thesequence in this part of the structure is not conserved across thefamily, forms very little regular secondary structure, and packsprimarily on the periphery of the molecule. Based on this analysis, itis expected that IL-17C and IL-17E which also possess an extra cysteinein strand 2 are also likely to have their N-termini in significantlydifferent conformations than that for IL-17F and IL-17. This analysisdivides the family into two classes based on the disulfide-bondingpattern of the N-terminus.

An impressive feature of the structure of IL-17F is the unusually largecavity formed by the residues in the dimer interface (FIG. 52) which issuggestive of a region that might bind another molecule. The cavity (twoper dimer) is composed of a combination of residues that are eitherstrictly conserved or always possess a similar chemical character (Tyr54, Tyr 63, Pro 64, Val 120), as well as others that are extremelyvariable among IL-17 family members (Arg 37, Val 38, Met 40, Ala 95)providing potential to impart specificity for intermolecularinteractions. The cavity does not have a pronounced electrostaticsurface feature, but instead is formed by a combination of hydrophobic,polar, and charged residues (see FIG. 51A and FIG. 52A). Based onsequence analysis, an analogous cavity would be expected to exist inother IL-17 family members; however given the likely differentconformation of the N-terminal extension, the specific characteristicsof the cavity could be quite different in IL-17B, IL-17C, and IL-17E.

NGF binds its high affinity receptor, TrkA, in a position analogous tothe location of the cavities in IL-17F. FIG. 52B and FIG. 52C showIL-17F and NGF in the same orientation highlighting the locations of thecavities and the TrkA binding sites (expected to be utilized by allneurotrophin/Trk complexes; Weismann et al. Nature 401:184-188 (1999)).The known structures of neurotrophin homodimers (NGF, NT3, NT4) alsohave an indentation on their surfaces at this position but it is muchsmaller and more modest than the cavity in IL-17F (McDonald et al.,Nature 354:411-414 (1991)); Butte et al., Biochemistry 37:16846-16852(1998); Robinson et al., Protein Sci. 8:2589-2597 (1999)). Trk familymembers are receptor tyrosine kinases that interact with neurotrophinsvia their membrane-proximal extracellular Ig-like domain. While it isnot expected that the structure of IL-17R or IL-17RH1 contains anIg-like fold, IL-17 proteins and neurotrophins could employ similarregions on their surfaces to bind their receptor.

Neurotrophins not only bind specific Trk receptors, but also can bindsimultaneously p75^(NTR) a second receptor common to all neurotrophins.p75^(NTR) binds its neurotrophin ligands via a cystine-richextracellular domain that is expected to resemble the structures oftumor necrosis factor receptor 1 (TNFR1) or death receptor 5 (Banner etal., Cell 73:431-445 (1993); Hymowitz et al., Mol. Cell 4:563-571(1999); Mongkolsapaya et al., Nat. Struc. Biol. 6:1048-1053 (1999)). Amodel of the NGF:p75^(NTR) interaction has been proposed based onmutagenesis data (Weismann, C., and de Vos, A. M., Cell. Mol. Life Sci.58:1-12 (2001)) and suggests that the loops at either end of the liganddimer as well as the flat surface on each protomer interact withp75^(NTR). The sequences of IL-17R and IL-17RH1 do not resemblep75^(NTR) and are not expected to adopt a TNFR1-like fold. However,given the similarity in IL-17 and neutrophin folds, it is reasonable toconsider the possibility of a second receptor component for IL-17s,analogous to the neutrophin system.

Further, the protein späztle has also been suggested to adopt aneurotrophin fold (Mizuguchi et al., TIBS 23:239-242 (1998)) and hasbeen shown genetically (although not by direct binding experiments) tobe a receptor for the drosphila Toll receptor (Morisato et al. Cell76:677-688 (1994)). Since IL-17 signals through NF-κB in a pathwaysimilar to that used by IL-1 and Toll receptors, which share a commonfold for their intracellular domain although their extracellular domainsare very different, it is reasonable to expect that either theintracellular or extracellular domains of IL-17 receptors, includingother as yet unknown components of the signalling complex, maystructurally resemble portions of these receptors. However, regardlessof receptor structure, the mode of interaction between IL-17 ligands andreceptors will most likely involve the deep cavities in the sides ofIL-17 dimer structure.

Example 35 mIL-17E Transgenic Mice—In Vivo Multi-Organ InflammatoryModel

Human IL-17E (PRO10272; SEQ ID NO:6) has been shown to be a potentinflammatory cytokine inducing the expression of a variety of otherchemokines and cytokines including IL-6, IL-8, G-CSF from cell linesderived from a variety of lineages and thereby acts to influenceinflammation and hematopoiesis. The authors herein have identified themurine ortholog of IL-17E (mIL-17E) and transgenic mice have beendeveloped in order to characterize its actions in vivo. The authors havealso identified the receptor for human IL-17E (IL-17RH1; also calledEvi27 or IL-17BR). Interestingly, the murine ortholog of IL-17RH1(designated herein as mIL-17ER) has been identified at Evi27, a commonsite for of retroviral integration in BXH2 murine leukemias. Theproviral integrations result in increased expression of the receptor andits role in myeloid leukemia and growth and/or differentiation ofhematopietic cells has also been suggested (see Tian, E., et al.,Oncogene 19:2098 (2000). In the present studies, the biologicalconsequences of overexpression of mIL-17E, both unique to IL-17E andsimilar to IL-17 (SEQ ID NO:40) were revealed, and these results aredescribed below.

A. Materials and Methods

Generation of mIL-17E Transgenic Mice

The cDNA encoding for the mature murine IL-17E protein (hereindesignated SEQ ID NO:41) with the putative signal sequence from humanIL-17E (PRO10272; SEQ ID NO:6) was cloned into a plasmid containing ratmyosin light chain promoter sequence followed by a sequence derived fromthe human growth hormone gene (hGH) including the 4^(th) and 5^(th)exons and 3′ UTR plus poly A to improve expression of the transgene (seeFaerman, A., and Shani, M., Development 118:919 (1993); and Shani, M. etal., Mol. Cell. Biol. 8:1006 (1998). The expression cassette fragmentwas excised and purified and injected into one-cell mouse eggs preparedfrom FVBXFVB matings. Genotyping was done by PCR analysis of the DNAfrom tail biopsies using primers against specific sequences in theexpression cassette. Expression levels of mIL-17E were determined byTaqman RT-PCR reactions (Perkin Elmer) on total RNA samples derived frommuscle biopsy.

Determination of Gene Expression

Total RNA samples from various mouse tissues were prepared using TRIZOLReagent according to manufacturer's instructions (GIBCO-BRL). The mRNAexpression levels for various cytokines, chemokines and adhesionmolecules and IL-17RH1 (IL-17E receptor) were determined by TaqmanRT-PCR (Perkin Elmer) using gene specific primers and probes. Expressionlevels of 18S gene were used as normalization control.

Histological Analysis

Routine necropsy was performed. Tissues for light microscopy werecollected and fixed overnight in 10% neutral buffered formalin, embeddedin paraffin, sectioned at 5 μm and stained with hematoxylin and eosin.Blood samples were collected and processed, and FACS analyses wereperformed on Epics XL-MCL (Coulter) using various antibodies (BDPharMingen) according to manufacturers' instructions.

Measurement of Serum Proteins

Serum IgG1 and IgG2a levels were assessed using a sandwich enzyme-linkedimmunosorbent assay (ELISA). Anti-mouse IgG1 and IgG2a coatingantibodies (PharMingen, San Diego, Calif.) were diluted to 1.0 and 2.5μg/ml in PBS (pH 7.2), respectively, and added to separate 96 wellsplates (Nunc Immuno Plate Maxisorp) then incubated overnight at 4° C.Plates were washed 3× (0.05% Tween-20) then blocked (0.5%

BSA in PBS) and incubated 2 hours at room temperature with gentleagitation then washed 3×. Mouse IgG1 standard (10 μg/ml standard stock,lot #31357-30A) was diluted to 25 ng/ml in assay buffer (0.5% BSA 0.05%Tween-20 in PBS) and 2-fold serial dilutions were performed to create a7 point standard curve ranging from 25-0.39 ng/ml. Mouse IgG2a standard(Southern Biotechnology, Birmingham, Ala.) was diluted to 400 ng/ml inassay buffer and 2-fold serial dilutions were performed to create a 7point standard curve ranging from 400-6.25 ng/ml. Serum samples wereserially diluted 2-fold in assay buffer to fall within the respectivestandard curve ranges. Standard or sample was added to each plate andincubated for 2 hours at room temperature with gentle agitation thenwashed 6×. Biotinylated anti-mouse IgG1 and IgG2a detection antibodies(PharMingen, San Diego, Calif.) were diluted 1:2000 in assay buffer andadded to each plate at 100 μl/well then incubated at room temperaturefor 1 hour with gentle agitation then washed 6×. Strepavidin-HRP (APBiotech, Piscataway, NJ) diluted 1:20,000 in assay buffer was added andincubated for 30 minutes at room temperature with gentle agitation thenwashed 6×. Tetramethyl benzidine (TMB) substrate solution (Kirkegaardand Perry, Gaithersburg, Md.) was added to each well and color wasallowed to develop for 4-6 minutes. Reaction was stopped with 1 Mphosphoric acid and absorbance was read at 450 nm. Serum IgE levels wereassessed by sandwich ELISA using Mouse IgE OptEIA kit (PharMingen, SanDiego, Calif.). Serum IL-5, IL-13, G-CSF, IFN-γ and TNF-α were measuredusing ELISA kits (R & D systems) according to manufacturers'instructions.

Recombinant Proteins and Cell Cultures

NIH-3T3 and ST2 cells were grown in HGDMEM with 10% heat activated FBS,2 mM L-glutamine, 1× Penicillin and Streptomycin. The cultures wereinitiated at 500,000/60 mm culture disk. All cultures were grown intriplicate. At 24 hours factors were added to the cultures; 4 hours and24 hours later conditioned media were removed and frozen for ELISA, andcells were lysed for RNA extraction in the dish using a Qiagen Rneasykit according to manufacturer's instructions. The recombinant IL-17 waspurchased (R & D systems) and IL-17E was prepared as previouslydescribed in EXAMPLE 3 above.

Statistical Analysis

For the body weights, hematologic analysis, and FACS on PBMC, thestatistical significance was determined by Student's T test. Geneexpression data were statistically analyzed by ANOVA using StatViewsoftware (Calabasas, Calif.). A value of p<0.05 was taken assignificant.

B. Results

Identification of the Murine Ortholog of IL-17E (mIL-17E)

Identification of mIL-17E was identified through sequence comparisonwith expressed sequence tag information (Accession number AI430337)present in Genbank. Several cDNA clones were subsequently isolated andthe longest cDNA clone encoded a partial signal sequence and thepredicted mature protein of murine IL-17E (mIL-17E) which was 85%identical to human IL-17E and 17-22% identical to other members of theIL-17 family (see FIG. 53). Attempts including 5′ racing, failed toidentify cDNA that contained an initial codon. Analysis of mRNAexpression in several mouse tissues indicated that IL-17E is expressedin brain, heart and testes, whereas little expression was detected inliver, lung or spleen (see FIG. 54). Tissue distribution of IL-17E inhuman tissues is also shown in FIG. 54 as well as in FIG. 23.

MIL-17E Transgenic Mice (TG) have Elevated Liver Enzymes, are Jaundicedand Growth Retarded

Transgenic mice have not been previously reported for any member of theIL-17 family. Attempts to generate transgenic mice that ubiquitouslyoverexpress murine IL-17 were unsuccessful (see Schwarzenberger, P., etal., J. Immunol. 163:6383 (1998)). The reason for this failure remainsunknown, but it is probable that overexpression of potentpro-inflammatory cytokines such as IL-17 during early development islethal. Thus, rat skeletal myosin light chain 2 promoter was chosen tooverexpress murine IL-17E in TG mice, since this promoter is known todirect a high level gene expression starting 6-9 days after birth,presumably giving rise to circulating mIL-17E (see Faerman, A., andShani, M. Development 118:919 (1993) and Shani, M., et al., Mol. CellBiol. 8:1006 (1998)). The mice were housed in a specific pathogen freeenvironment and multiple founders were analyzed. All TG pups weresignificantly smaller than their non-TG littermates by 9 days of age(p<0.05). This difference in body weights was retained at 21, 28, 42,56, and 98 days of age, suggesting that the transgenic mice were growthretarded (FIG. 55).

At 6 weeks of age, most of the TG founder mice were jaundiced (FIG. 56),indicating bilirubin deposition in the tissues. Consistent with this,serum levels of bilirubin were significantly elevated in the mIL-17E TGmice (by 50-100 fold, FIG. 57). In addition, serum levels of liverenzymes were markedly elevated, suggestive of liver damage in themIL-17E TG mice (FIG. 57).

Over-Expression of mIL-17E in TG Mice Induces Gene Expression ofCytokines in Multiple Tissues

The in vivo consequences of the overexpressed mIL-17E were studied byexamining the gene profilesof both Th1 and Th2 cytokines. Human IL-17Ereceptor (designated IL-17RH1), is expressed in multiple tissues,especially in liver and kidneys with lower abundance in testes, brainand small intestine and other tissues (FIGS. 31A-31B). Similarexpression patterns for the mIL-17E receptor were also observed in themouse wherein expression was found to be especially abundant in liverand kidneys (see Shi, Y., et al., J. Biol. Chem. 275:19167 (2000) andLee, J., et al., J. Biol. Chem. 276:1660 (2001)).

In view of these observations, expression of inflammatory cytokines inthese tissues was measured using quantitative RT-PCR assays [relativeexpression TG versus Non-TG]. The transcripts for Th2 cytokines IL-4 andIL-10 were found to be significantly induced in liver from TG mice (FIG.58A). IL-4, IL-10 and IL-13 were also significantly induced in kidneysfrom TG mice (FIG. 58B—top panel). Interestingly, some of thesecytokines were also dramatically induced in lungs (IL-4 and IL-10, FIG.58C—bottom panel), heart (IL-10, and IL-13, results not shown), spleen(IL-4, IL-6 and IL-13, FIG. 58D—top panel) and intestines (IL-4, IL-5,IL-9 and IL-10, FIG. 58E—bottom panel), where normally very lowabundance of mIL-17ER mRNA in these tissues have been detected (Shi, Y.,et al., J. Biol. Chem. 275:19167 (2000) and Lee, J., et al., J. Biol.Chem. 276:1660 (2001)). These findings suggest that these tissues mayalso be responsive to IL-17E. As a consequence of these findings,mIL-17ER expression was measured in these other tissues in TG mice. FIG.59 demonstrates the up-regulation of IL-17E receptor expression inIL-17E transgenic mice in lung, kidney, liver, spleen and heart tissues(relative expression of non-TG and TG mice). As demonstrated in FIG. 59,mIL-17E receptor mRNA was substantially increased in multiple tissues,especially heart and lung (increased by 67- and 19-fold, respectively),consistent with the role of IL-17E in enhancing signaling in peripheraltissues by up-regulation of its own receptor. These cytokine profilessuggest that IL-17E may drive a Th2-like response. However, when thegene expression levels of Thl cytokines were measured (e.g.,interferon-(and TNF-α), elevated levels of these messages were alsoobserved in several tissues (FIGS. 58A-58E). Thus, the inflammatoryresponse induced by mIL-17E may not be strictly Th2 in character.

Murine IL-17E (mIL-17E) TG Mice have Increased Serum IL-13 and IL-5 andCirculating IgE and IgG1

To determine if the elevated gene expression seen above gave rise tocirculating cytokines and further affected antibody generation, serumlevels of several cytokines and antibodies were measured using specificELISAs. Both Th2 cytokines, IL-13 and IL-5 were increased in mIL-17E TGmice, however, serum TNF-α was also induced in the TG mice (FIG. 60). Inaddition, both serum IgE and IgG1 (characteristic of Th2 response) wereobserved to be significantly elevated in TG mice, but serum levels ofIgG2a (Th1 in character) were not altered (FIG. 61). These findingssuggest that IL-17E induces a systemic Th2-biased response.

Overexpression of mIL-17E Causes Neutrophilia and Eosinophilia inTransgenic Mice

In vivo expression of IL-17 via adenoviral-mediated delivery causesneutrophilia in mice, but no effect on eosinophils has been reported(see Schwarzenberger, P., et al., J. Immunol. 161:6383 (1998)). A studywas undertaken to determine whether overexpression of mIL-17E had asimilar effect. FACS analyses of peripheral blood mononuclear cells(PBMC) were performed using specific cell surface markers. CD3⁺ T cellsor CD19⁺ B cells were found to be significantly reduced in TG mice,compared to those of non-TG mice (FIG. 62). However, there was no changein the CD4⁺/CD8⁺ ratio (results not shown). When PBMC were stained forGR-1⁺ neutrophils, TG mice had significantly increased neutrophils asdemonstrated in FIG. 62. Consistent with these findings, the absolutecell counts of neutrophils were increased by 8-10-fold in TG mice. Asshown in FIG. 63, the absolute counts of eosinophils were alsosignificantly increased but the absolute number of lymphocytes wasslightly reduced. FACS analyses of cells isolated from spleen and lymphnodes also showed significantly increased neutrophils in the TG mice(data not shown). These findings indicate that IL-17E stimulateshematopoiesis and causes neutrophilia and eosinophilia in vivo. As shownbelow, these effects may be mediated in part by increased IL-5 andG-CSF.

Murine IL-17E Induces Expression of Neutrophil-Specific Chemokine GROaand Adhesion Molecules

Like IL-17, IL-17E stimulates IL-8 production in cell cultures (shown inFIG. 34; see also Fossiez, F., et al., Int. Rev. Immunol. 16:541 (1998),and Lee, J., et al., J. Biol. Chem. 276: 1660 (2001). In order to seethe effect of mIL-17E on the gene expression of other chemokines andadhesion molecules in vivo that might contribute to the immuneinfiltrate in tissues, mRNA levels were examined for GRO∀, MCP-1, ICAM-1and VCAM-1 in multiple tissues from the TG mice. The GROα mRNA wassignificantly induced in liver, kidneys, lungs and heart, while ICAM-1was increased in the liver and VCAM-1 in kidney (see FIGS. 58A-58E).These findings suggest that mIL-17E may induce production of chemokinesand adhesion molecules in epithelial, endothelial cells and fibroblastsin various tissues, contributing to the recruitment of neutrophils,lymphocytes and other infiltrating cells.

IL-17E Stimulates G-CSF Production

IL-17 stimulates production of G-CSF, a potent inducer ofgranulopoiesis, in vivo and from stromal cells in vitro (Fossiez, F., etal., J. Exp. Med. 183:2596 (1996); Schwarzenberger, P., et al., J.Immunol. 161:6383 (1998); and Metcalf, D., Science 254:529 (1991)). Todetermine if G-CSF was induced by IL-17E in vivo, G-CSF mRNA levels inTG tissues were measured. G-CSF mRNA levels were markedly increased inliver, kidneys and spleen (FIGS. 58A-58E and data not shown). Consistentwith this, serum G-CSF was also dramatically induced in the TG mice(FIG. 64). IL-17 directly stimulates G-CSF production in stromal cellsNIH3T3 and ST2 (Fossiez, F., et al., J. Exp. Med. 183:2596 (1996)). Todetermine whether IL-17E has a similar activity, NIH3T3 and ST2 weretreated with recombinant IL-17E. Like IL-17, IL-17E stimulatedproduction of G-CSF (FIG. 65). These findings suggest that IL-17Einduces G-CSF production and the increased G-CSF may contribute to thegranulopoiesis seen in the mIL-17E TG mice.

Overexpression of mIL-17E Causes Multi-Organ Inflammation

A comprehensive histological tissue survey showed that mIL-17Etransgenic mice had chronic inflammation in multiple tissues. Tissuesconsistently affected include liver, heart, lungs, lymph node, kidneys(renal pelvis and mild glomerular changes), spleen and urinary bladder.Inflammation in these tissues was comprised of mixed infiltrates ofneutrophils, eosinophils, lymphocytes, plasma cells and macrophages. Inthe liver, mIL-17E transgenic mice evaluated had severecholangiohepatitis with adenomatous hyperplasia of bile ducts,periportal fibrosis, and variable oval cell hyperplasia (FIG. 66B versusFIG. 66A). Special stains including Warthin Starry and PAS were negativefor Helicobacter sp. and fungal elements, respectively (not shown). Inthe lungs, mIL-17E transgenic mice consistently develop diffuseinterstitial and peribronchial inflammation with more severe changes inthe highest expressing founders (FIGS. 66C-66D). In addition to themixed inflammatory cell infiltrate discussed above, alveolar spaces werefilled with numerous macrophages that were occasionally multinucleatedand often distended with long, thin, cytoplasmic crystals, similar tothose reported in the lungs of other mutant mouse models that haveeosinophilic inflammation. These histological findings suggestubiquitous expression of mIL-17E causes profound pathologic changes inmultiple organs.

C. Discussion

The biological consequences of IL-17E exposure have both interestingsimilarities and clear distinctions to those reported for IL-17. LikeIL-17, IL-17E impact diverse tissues. This reflects, in part, the broadexpression of its receptor (Shi, Y., et al., J. Biol. Chem. 275:19167(2000); Lee, J., et al., J. Biol. Chem. 276:1660 (2001); and Yao, Z., etal., Immunity 3:811 (1995)). IL-17E promotes substantial neutrophilia, aresponse that may be due to the observed induction of G-CSF. IL-17 hasalso been shown to induce production of G-CSF and promote neutrophilia(Fossiez, F., et al., J. Exp. Med., 183:2596 (1996); Schwartzenberger,P., et al., J. Immunol. 161:6383 (1998); and Metcalf, D., Science254:529 (1991)). Similarly, IL-17 and IL-17E induce local production ofchemokines which target neutrophils such as GROV and IL-8 (Lee, J., etal., J. Biol. Chem 276:1660 (2001); and Witowski, J., et al., J.Immunol. 165:5814 (2000)). Furthermore, IL-17 and IL-17E are associatedwith increased expression of ICAM-1 and other inflammatory cytokines(Fossiez, F., et al., Int. Rev. Immunol. 16:541 (1998) and Faerman, A.,and Shani, M., Development 118:919 (1993)). In contrast, IL-17Eoverexpression resulted in the promotion of a systemic Th2-biased immuneresponse. This response has not been noted with chromic IL-17 exposure(Schwarzenberger, P., et al., J. Immunol. 161:6383 (1998)). The Th2feature of this response was characterized by cytokine profile, thepresence of increased serum IgE and IgG1, and an increase in eosinophilnumbers. It should be noted that in vivo expression of IL-17 increasedperipheral white blood count and 2-fold increases in lymphocytes(Schwarzenberger, P., et al., J. Immunol. 164:4783 (2000), andSchwarzenberger, P., et al., J. Immunol. 161:6383 (1998)). In contrast,mIL-17E transgenic mice appeared to be slightly lymphopenic.

Long-term exposure to IL-17E causes multi-organ inflammation. Theinflammatory infiltrate in IL-17E TG mice is comprised of eosinophils;however, mixed cellular infiltrates including neutrophils andlymphocytes are frequently present and may result from secondarynecrosis or induction of proinflammatory chemokines. Epithelialhyperplasia was observed in multiple tissues. Interestingly, IL-17ERH1message is elevated in multiple tissues in the transgenic mice,suggesting that the spectrum of tissues upon which IL-17E can act isinfluenced by the regulation of receptor expression.

Although overexpression of IL-17E appears to be drive a Th2-biasedresponse, it should be noted that increased expression of several Thlcytokines (IFN-(mRNA and serum TNF-α) were also observed in the TG mice.This might have been from secondary tissue necrosis and contributed tothe tissue-specifc variation in immunologic response and pathologicalchanges. Systemic exposure to IL-17E elicits inflammation in multipleorgans, however, it is conceivable that local expression of IL-17E intissues caused by certain disease conditions may induce a morelocalized, tissue-specific immunologic response and pathologic changes.Identification of such disease conditions certainly will providetherapeutic opportunities.

In summary, the authors have developed a transgenic mouse modeloverexpressing murine IL-17E under the control of the muscle myosinlight chain 2 gene (MLCH) promoter. The systemic overexpression ofmIL-17E upregulates gene expression of Th2 cytokines, including IL-4,IL-10 and IL-13 in many tissues. Serum levels of IL-13 and IL-5 as wellas circulating IgE and IgG1 are also increased in transgenic mice.Furthermore, these profound immunological changes in mIL-17E transgenic(TG) mice are associated with pathological changes in multiple tissues,characterized by a mixed immune infiltration and epithelial cellhyperplasia. Taken together, these findings suggest that IL-17E is aunique pleiotrophic cytokine and may be an important mediator ofinflammatory and immune responses.

Example 36 Microarray Analysis to Detect Overexpression of PROPolypeptides in Cancerous Tumors

Nucleic acid microarrays, often containing thousands of gene sequences,are useful for identifying differentially expressed genes in diseasedtissues as compared to their normal counterparts. Using nucleic acidmicroarrays, test and control mRNA samples from test and control tissuesamples are reverse transcribed and labeled to generate cDNA probes. ThecDNA probes are then hybridized to an array of nucleic acids immobilizedon a solid support. The array is configured such that the sequence andposition of each member of the array is known. For example, a selectionof genes known to be expressed in certain disease states may be arrayedon a solid support. Hybridization of a labeled probe with a particulararray member indicates that the sample from which the probe was derivedexpresses that gene. If the hybridization signal of a probe from a test(disease tissue) sample is greater than hybridization signal of a probefrom a control (normal tissue) sample, the gene or genes overexpressedin the disease tissue are identified. The implication of this result isthat an overexpressed protein in a diseased tissue is useful not only asa diagnostic marker for the presence of the disease condition, but alsoas a therapeutic target for treatment of the disease condition.

The methodology of hybridization of nucleic acids and microarraytechnology is well known in the art. In the present example, thespecific preparation of nucleic acids for hybridization and probes,slides, and hybridization conditions are all detailed in U.S.Provisional Patent Application Ser. No. 60/193,767, filed on Mar. 31,2000 and which is herein incorporated by reference.

In the present example, cancerous tumors derived from various humantissues were studied for PRO polypeptide-encoding gene expressionrelative to non-cancerous human tissue in an attempt to identify thosePRO polypeptides which are overexpressed in cancerous tumors. Two setsof experimental data were generated. In one set, cancerous human colontumor tissue and matched non-cancerous human colon tumor tissue from thesame patient (“matched colon control”) were obtained and analyzed forPRO polypeptide expression using the above described microarraytechnology. In the second set of data, cancerous human tumor tissue fromany of a variety of different human tumors was obtained and compared toa “universal” epithelial control sample which was prepared by poolingnon-cancerous human tissues of epithelial origin, including liver,kidney, and lung. mRNA isolated from the pooled tissues represents amixture of expressed gene products from these different tissues.Microarray hybridization experiments using the pooled control samplesgenerated a linear plot in a 2-color analysis. The slope of the linegenerated in a 2-color analysis was then used to normalize the ratios of(test:control detection) within each experiment. The normalized ratiosfrom various experiments were then compared and used to identifyclustering of gene expression. Thus, the pooled “universal control”sample not only allowed effective relative gene expressiondeterminations in a simple 2-sample comparison, it also allowedmulti-sample comparisons across several experiments.

In the present experiments, nucleic acid probes derived from the hereindescribed PRO polypeptide-encoding nucleic acid sequences were used inthe creation of the microarray and RNA from the tumor tissues listedabove were used for the hybridization thereto. A value based upon thenormalized ratio:experimental ratio was designated as a “cutoff ratio”.Only values that were above this cutoff ratio were determined to besignificant. Table 9 below shows the results of these experiments,demonstrating that various PRO polypeptides of the present invention aresignificantly overexpressed in various human tumor tissues as comparedto a non-cancerous human tissue control. As described above, these datademonstrate that the PRO polypeptides of the present invention areuseful not only as diagnostic markers for the presence of one or morecancerous tumors, but also serve as therapeutic targets for thetreatment of those tumors.

TABLE 9 Molecule is overexpressed in: as compared to: PRO1031 lung tumoruniversal normal control PRO1122 breast tumor universal normal controlPRO1122 lung tumor universal normal control PRO5801 colon tumoruniversal normal control PRO21175 breast tumor universal normal controlPRO21175 colon tumor universal normal control PRO21175 lung tumoruniversal normal control

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,USA (ATCC):

Material ATCC Dep. No. Deposit Date DNA59294-1381 209866 May 14, 1998DNA62377-1381-1 203552 Dec. 22, 1998 DNA147531-2821 PTA-1185 Jan. 11,2000 DNA173894-2947 PTA-2108 Jun. 20, 2000 DNA115291-2681 PTA-202 Jun.8, 1999 DNA164625-2890 PTA-1535 Mar. 21, 2000 DNA119502-2789 PTA-1082Dec. 22, 1999 DNA154095-2998 PTA-2591 Oct. 10, 2000

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposits will be made availableby ATCC under the terms of the Budapest Treaty, and subject to anagreement between Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC § 122 and the Commissioner's rules pursuantthereto (including 37 CFR § 1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

What is claimed is:
 1. A method for detecting the presence of tumor inan mammal, said method comprising comparing the level of expression of(i) the polypeptide having the amino acid sequence of SEQ ID NO:18; or(ii) the polypeptide encoded by the the full-length coding sequence ofthe cDNA deposited under ATCC accession number PTA-2591 in (a) a testsample of cells taken from said mammal and (b) a control sample ofnormal cells of the same cell type, wherein a higher level of expressionof said polypeptide in the test sample as compared to the control sampleis indicative of the presence of tumor in said mammal.
 2. The method ofclaim 1, wherein said tumor is kidney tumor, lung tumor, or rectaltumor.